Nanopore platform for DNA/RNA oligo detection using an osmium tagged complementary probe

ABSTRACT

Provided for herein is a method for detecting the presence of a nucleic acid target molecule in a biological sample. In certain aspects, the method comprises contacting a test sample that comprises (i) a biological sample comprising a nucleic acid target molecule and (ii) an osmylated single-stranded oligonucleotide probe comprising at least one pyrimidine residue covalently bonded to a substituted or unsubstituted Osmium tetroxide (OsO4)-2,2′-bypyridine group (OsBp group).

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with government support under grant #R43HG010841awarded by the NIH/NHGRI, SBIR program. The government has certainrights in the invention.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing in ASCIItext file (Name 68812_199738_ST25.txt; Size: 6755 bytes; and Date ofCreation: Oct. 2, 2020) filed with the application is incorporatedherein by reference in its entirety.

BACKGROUND

Blood draw or other body fluid samples, known as liquid biopsies(Bronkhorst, A. J., Ungerer, V., & Holdenrieder, S. (2019); Vidal, J.,Taus, A., & Montagut, C. (2020); Pös, O., Biró, O., Szemes, T., & Nagy,B. (2018); Oellerich, M., Schütz, E., Beck, J., & Walson, P. D. (2019);Stewart, C. M., & Tsui, D. (2018); Giannopoulou, L., Kasimir-Bauer, S.,& Lianidou, E. S. (2018); Satyal, U., Srivastava, A., & Abbosh, P. H.(2019); Finotti, A. et al. (2018); Valpione, S., & Campana, L. (2019);and Kwapisz D. (2017)) contain pertinent information regarding thehealth status of an individual, the progress of a disease, whether ornot an individual is disease-free after surgery, and even whether or nota certain therapy strategy seems promising (Vidal, J., Taus, A., &Montagut, C. (2020)). Liquid biopsies are far less invasive proceduresthan surgical/tumor biopsy. Body fluids contain cell-free DNA (cfDNA), afraction of which can originate from a tumor (circulating tumor DNA;ctDNA). In 2016 the US Food and Drug Administration approved the firstliquid biopsy test for EGFR-activating mutations in patients withnon-small-cell lung cancer as a companion diagnostic test to enabletherapy selection (Kwapisz D. (2017)). Cell-free DNA is believed to befragmented and shorter in diseased individuals compared to healthy (Pös,O., Biró, O., Szemes, T., & Nagy, B. (2018)). In addition to DNA, bodyfluids contain transfer RNA-derived fragments and non-coding RNA oligosin the range of 20 to 300 nucleotides (nt) (Kim H K, Yeom J H, Kay M A.(2020); Poller W, et. al. (2018); Mitchell, P. et al. (2008); Aggarwal,V., Priyanka, K., & Tuli, H. S. (2020); Meseure, D., Drak Alsibai, K.,Nicolas, A., Bieche, I. & Morillon, (2015)). Among them is a group ofsingle-stranded (ss) RNAs, 17 to 25 nt long, known as microRNAs ormiRNAs. They were discovered 20 years ago and proven to be the tinyregulators that control the post-transcriptional expression of proteins(Ambros, V. (2001); Bartel, D. P. (2004)). miRNAs are highly conservedand surprisingly stable in body fluids (Mitchell, P. et al. (2008);Mall, C., Rocke, D. M., Durbin-Johnson, B., & Weiss, R. H. (2013)).Currently there are over 2,300 human miRNAs known (Alles J. et al.(2019)), the subject of over 100,000 scientific publications. Up- ordown-regulation of miRNAs is associated with various human diseasesincluding cancer, heart disease, kidney disease, obesity, diabetes, etc.(Bartel, D. P. (2004)); miRNAs are proposed as biomarkers (Farazi, T.A., Hoell, J. I., Morozov, P., & Tuschl, T. (2013); Pogribny, I. P.(2017)) and as potential therapeutics (Rupaimoole, R. & Slack, F. J.(2017)) in personalized medicine. Body fluids contain trace amounts ofcfDNA, ctDNA, fragmented coding RNAs (Kim H K, Yeom J H, Kay M A.(2020)), non-coding RNAs (Poller W, et. al. (2018)), and miRNAs, thatrequire simple, validated, and highly sensitive assays for testing(Finotti, (2018); Valpione, S., & Campana, L. (2019); Raabe, C., TangT., Brosius J., & Rozhdestvensky, T. (2014)). Current technologies for“small RNA” identification and quantitation, include microarray, NGSsequencing (Ion Torrent or Illumina (small RNA-seq)), and qRT-PCR-basedmethods that have been employed so far with great success (Ferracin, M.,& Negrini, M. (2018); Valihrach, L., Androvic, P., & Kubista, M. (2020);Gines, G., Menezes, R., Xiao, W., Rondelez, Y., Taly, V. (2020)). Thesetechnologies, however, require substantial infrastructure and skilledpersonnel, are not well-suited for point-of-care testing, and are out ofthe question for home testing.

The last 30 years have seen a surge in nanopore-based technologies usingeither solid-state or protein nanopores (Kasianowicz, J. J., Brandin,E., Branton, D. & Deamer, D. W. (1996); Butler, T. Z., Gundlach, J. H. &Troll, M. (2007); Maglia, G., Heron, A. J., Stoddart, D., Japrung, D. &Bayley, H. (2010); Hague, F., Li, J., Wu, H. C., Liang, X. J. & Guo, P.S (2013); Fuller C. W. et al. (2016); Laszlo, A. H. et al. (2014);Oxford Nanopore Technologies website: nanoporetech.com, underResources/Publications). In 2014 Oxford Nanopore Technologies (ONT)introduced and commercialized the first portable nanopore device tosequence DNA and RNA practically anywhere, as long as a computer andinternet are available (Oxford Nanopore Technologies website:nanoporetech.com, under Resources/Publications). The ONT technology isbased on the CsGg protein nanopore (Cao, B. et al. (2014)), with a sub 2nm diameter, inserted in a planar lipid bilayer membrane that separatestwo electrolyte filled compartments (FIG. 1A). Applying a voltage acrossthe two compartments leads to a constant flow of electrolyte ions(I_(o)) via the pore, recorded as a function of time (i-t). The passageof a single molecule through the pore reduces I_(o) to a lower level ofresidual ion current (I_(r)). This is recorded as an “event” with(I_(r)) and residence time (τ) (FIG. 1B). Currently the ONT platform isexclusively used for DNA/RNA sequencing (Oxford Nanopore Technologieswebsite: nanoporetech.com, under Resources/Publications), whilecomparable nanopore platforms are successfully employed for singlemolecule analyses (Chen, X., Wang, L., & Lou, J. (2020); Chaudhary, V.,Jangra, S. & Yadav, N. R. (2018); Wanunu M, Dadosh T, Ray V, Jin J,McReynolds L, Drndić M. (2010); Gu, L. Q. & Wang Y. (2013); Arata, H.,Hosokawa, K., & Maeda, M. (2014); Henley, R. Y., Vazquez-Pagan, A. G.,Johnson, M., Kanavarioti, A. & Wanunu, M. (2015); Xi, D. et al. (2016);Zahid, 0. K., Wang, F., Ruzicka, J. A., Taylor, E. W. & Hall, A. R.(2016); Ding, Y. & Kanavarioti, A. (2016); Tian, K., Shi, R., Gu, A.,Pennella, M., & Gu, L. Q. (2017); Zhang, Y., Rana, A., Stratton, Y.,Czyzyk-Krzeska, M. F., & Esfandiari, L. (2017); Huang, G., Willems, K.,Soskine, M., Wloka, C. & Maglia, G. (2017); Galenkamp, N. S., Soskine,M., Hermans, J., Wloka, C. & Maglia, G. (2018); Sultan M., Kanavarioti,A. (2019); Cao, C. et al. (2019); Hao, W., Haoran T., Cheng Y., &Yongxin, L. (2019)).

ONT provides portable nanopore devices that carry two types of flowcells; the MinION with 512 channels and the Flongle with 126 channels,all monitored simultaneously (Oxford Nanopore Technologies website:nanoporetech.com, under Resources/Publications). ONT promotes thesedevices for direct sequencing of DNA and RNA with a minimum length of200 nucleotides (nt) (Oxford Nanopore Technologies website:nanoporetech.com, under Resources/Publications). To date, however,attempts to sequence RNAs shorter than 200 nt following ONT protocolappear unsuccessful (Workman, R. E. et al. (2019)), absentcircularization and rolling circle amplification to produce long DNAwith 100 nt repeats (Wilson, B. D., Eisenstein, M. & Soh, H. T. (2019)).Most of the DNA/RNA available in biological fluids is fragmented withlengths estimated in the range of 200 bp (Pös, O., Biró, O., Szemes, T.,& Nagy, B. (2018)) and miRNAs are very short (Ambros, V. (2001)), andtherefore not amenable to direct ONT sequencing protocols. Althoughseveral other experimental nanopore platforms have been successfullyused for miRNA profiling, they have not been shown as suitable forcommercial utilization. Thus, there remains a need for a technology thatis accessible, ready-to-use, relatively inexpensive, and does notrequire any special skills or infrastructure.

SUMMARY

Provided for herein is a method for detecting the presence of a nucleicacid target molecule in a biological sample. In certain aspects, themethod comprises the steps of: (a) contacting a test sample thatcomprises (i) a biological sample comprising a nucleic acid targetmolecule and (ii) an osmylated single-stranded oligonucleotide probecomprising at least one pyrimidine residue covalently bonded to asubstituted or unsubstituted Osmium tetroxide (OsO4)-2,2′-bypyridinegroup (OsBp group), wherein the sequence of the probe is at leastpartially complementary to the sequence of the nucleic acid targetmolecule, to allow the formation of a hybridized probe/target complex;(b) using a nanopore device to detect in the test sample the number ofevents wherein unhybridized osmylated-probe traverses the nanopore; and(c)(i) comparing the number of events detected in the test sample to anumber of corresponding probe sample events wherein unhybridizedosmylated-probe traverses the nanopore in the absence of the nucleicacid target, wherein a reduction in the number of events detected in thetest sample relative to the number of probe sample events is indicativeof the formation of the hybridized probe/target complex in step (a) andthe presence of the nucleic acid target molecule in the test sample;(c)(ii) comparing the number of events detected in the test sample tothe noise of a corresponding baseline sample that does not contain anyosmylated-probe, wherein an absence of an increase in the number ofevents detected in the test sample relative to the noise of the baselinesample is indicative of the formation of the hybridized probe/targetcomplex in step (a) and the presence of the nucleic acid molecule in thetest sample; and/or (c)(iii) comparing the number of events detected inthe test sample to a number of corresponding control sample eventswherein unhybridized osmylated-probe traverses the nanopore in thepresence of a known amount of the nucleic acid target molecule, whereina reduction in the number of events detected in the test sample relativeto the number of control sample events is indicative of increasedformation of the hybridized probe/target complex in step (a) and thepresence of a higher amount of the nucleic acid target molecule in thetest sample over the control sample or wherein an increase in the numberof events detected in the test sample relative to the number of controlsample events using the same amount of probe is indicative moreunhybridized probe and thus of a lower amount of the nucleic acid targetmolecule in the test sample compared to the control sample. In certainaspects, at least one osmylated pyrimidine is a thymine residue (T).

Further, certain aspects provide for a kit comprising an osmylated probeof this disclosure and a control nucleic acid target molecule that canhybridize to the probe and for the use of such probe for detection of anucleic acid target molecule with a nanopore device, wherein the nucleicacid target molecule is optionally a ctDNA, cfDNA, miRNA, or anon-coding RNA, optionally, wherein the non-coding RNA is less thanabout 300 bases long.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1A-D. (FIG. 1A) Schematic representation of a nanopore within aplanar bilayer lipid membrane that separates two electrolyte filledcompartments. Applying a constant voltage to the flow cell guides thepassage of ions through the nanopore creating a measurable ioniccurrent. (FIG. 1B) The i-t trace obtained from a voltage-drivenion-channel experiment where the constant flow of electrolyte ions(I_(o)) via the pore is interrupted by the passage of molecules. Thesemolecules appear as “events” with residual ion current I_(r) andresidence time τ. (FIG. 1C) OsBp labeling reaction: OsO₄ and2,2-bipyridine (bipy) have a low association constant, but their mixtureadds to the C5-C6 double bond of pyrimidines and forms a stableconjugate. The addition of OsBp creates a chromophore that absorbs inthe range of 312 nm where native nucleic acids do not absorb (seeExamples). (FIG. 1D) Illustration of the concept behind the proposeddiagnostic test. ssDNA and ssRNA traverse the nanopore and exhibit fewcounts because they traverse faster, compared to the device's relativelyslow acquisition rate; ds nucleic acids are too big and do not traversethis nanopore. Despite being bulkier than ss native nucleic acids,osmylated ss nucleic acids traverse the pore, but more slowly comparedto the device's acquisition rate and consequently produce numerousevents. When an osmylated nucleic acid (probe) is added to a sample thatcontains its complementary nucleic acid (target), the probe and thetarget form a hybrid. When the target's concentration is equal or higherthan the probe's concentration, the probe is hybridized. The probe isthen prevented from traversing the pore, and few or no events areobserved. The absence of target in the sample is evidenced by thenumerous events produced by the probe while it freely traverses thepore.

FIG. 2A,B. Voltage-driven ion-channel (nanopore) experiments conductedwith the Flongle ONT device; samples in >90% ONT buffer. (FIG. 2A) 1 hat −200 mV using the same Flongle flow cell: (i) 5 μM probe T8(RNA) and(ii) 5 μM each a mixture of T8(RNA) and d(CT)₁₀. It is noticeable thatthese two molecules are only partially complementary to each other.Count of events (counts) were obtained using the OsBp_detect software toanalyze and report the raw fast-5 file data acquired with MINKNOW(Kanavarioti, A., & Kang, A. See RNA(OsBp) event detection Pythonpackage in a public repository: on the world wide web atgithub.com/kangaroo96/osbp_detect and for step-by-step installationinstructions see at the world wide web atgithub.com/kangaroo96/osbp_detect/blob/master/instructions.md; seeExamples). Counts were plotted as a function of I_(r)/I_(o) with a binsize of 0.05. (FIG. 2B) 2 h at −190 mV using the same Flongle flow cell:(i) 504 probe dmiR122, (ii) 504 each a mixture of dmiR122 and miRNA122,and (iii) 1 h at −180 mV a mixture 10 μM each of miRNA 122 and miRNA140using a different Flongle flow cell. It is noticeable that dmiR122carries 4 OsBp moieties and its sequence is perfectly complementary tomiRNA122. Data acquisition and analysis as described under (FIG. 2A).

FIG. 3A-D. Alternative approaches to testing hybridization betweenosmylated probes and targets. (FIG. 3A) Enzymatic elongation ofosmylated primers using ssM13mp18 DNA as the template and DNApolymerase; time points obtained at 5, 10 and 20 min. No primer andM13rev(−48) used as negative controls. With the exception of BJ1, allthe other osmylated primers exhibit enzymatic elongation comparable tothe positive control M13fwd(6097). Absence of elongation with BJ1 isattributed to the presence of a T(OsBp) at the 3′-end. (FIG. 3B), (FIG.3C) and (FIG. 3D) Overlapping HPLC profiles from the analyses ofdifferent samples, with samples at about 504 in about 90% ONT buffer.The same HPLC method B was used for all the samples (see Examples).Intact ss oligos and ds oligos appear as sharp peaks, whereas osmylatedoligos and hybrids with one osmylated strand appear as broad peaks;hybrids elute later compared to ss nucleic acids. (FIG. 3B) Samplecomposition: intact BJ2, intact complement of primerM13 for(−41). TheHPLC profile of their equimolar mixture is consistent withhybridization. (FIG. 3C) Sample composition: miRNA21, probe 21EXTcarrying 8 dT(OsBp) moieties, and equimolar mixture of the two. HPLCprofile of the mixture consistent with NO hybridization, attributed tothe high number of single OsBp tags, 6 within a sequence of 22 nt,likely to distort the helical structure of the probe, and prevent dsformation. (FIG. 3D) Sample composition practically the same as under(FIG. 3B), with the exception that in these samples BJ2 carries 6dT(OsBp) moieties (first peak with broad shape and absorbance at 312nm). HPLC profile of the mixture is consistent with hybridization,attributed to the fact that most of the OsBp moieties are adjacent, sothat the rest of the sequence can still hybridize with the target.

FIG. 4A-D. Same samples tested by HPLC and by nanopore; samples in >90%ONT buffer. (FIG. 4A) Overlapping HPLC profiles of samples, at about 0.2nmoles, in ONT buffer (i) probe BJ1, (ii) target is shown here at a muchhigher load, and (iii) mixture of probe at about 0.2 nmoles and targetwith about 30% excess over the probe (hybrid). The HPLC profile of themixture is consistent with hybridization. (FIG. 4C) Overlapping HPLCprofiles (i) of the probe BJ2 TA(OMe) at about 0.2 nmole load and (ii)its approximately equimolar mixture with the complementary target,complementary primerM13 for(−41). Target identified as a small peak, at5% excess over the probe. The HPLC profile of the mixture (hybrid) isconsistent with ds formation. HPLC profiles for these two samples areshown at both the 272 and 312 nm (see Examples). (FIG. 4B) and (FIG. 4D)One hour long nanopore experiments conducted with a MinION flow cellusing the same samples as in (FIG. 4A) and (FIG. 4C), respectively;Samples in (FIG. 4B) were used as is, while samples in (FIG. 4D) wereused after a 1000-fold or 100-fold dilution in ONT buffer. (FIG. 4B)Probe BJ1 was tested at −180 mV (dashed trace) and showed few counts. Nosample was added, the voltage was raised to −220 mV and an additionalnanopore experiment was conducted at −220 mV (solid trace) with countsexceeding 100,000. In contrast to the counts obtained with BJ1, thehybrid sample exhibited remarkably few counts (solid trace, every closeto the x-axis, tested 1 h at −190 mV). (FIG. 4D) Nanopore experimentsusing the same flow cell: (i) control/buffer test (0.75 h tested at −180mV), (ii) probe BJ2 TA(OMe) at 0.38 pmole load (2 h tested at −210 mV),(iii) equimolar mixture of probe and target at 3.8 pmole load each (1 htested at −210 mV). The observation of lower counts with the hybridsample compared to the probe sample suggest target identification foreither (FIG. 4B) and (FIG. 4D), in agreement with the HPLC results. Thenanopore experiment with BJ2 TA(OMe) probe at the 0.38 pmole loadsuggests probe detectability at the sub-pmole level. The experiment withthe hybrid sample in (d) indicates that the hybrid endures under theexperimental conditions of duration and applied voltage. Dataacquisition and analysis as described under FIG. 2A.

FIG. 5A-D. Nanopore experiments testing advanced probe designs and probedetection in 15% human serum-85% ONT buffer. (FIG. 5A) Three consecutivenanopore experiments conducted with the same flow cell. (i) Buffer test0.75 h at −180 mV, (ii) probe 2XdmiR122 tested 1.5 h at −180 mV (dashedline) and (iii) no sample added, voltage raised to −220 mV andexperiment run for 1.5 h. This set of experiments provided solidevidence that, at least, this probe is not traversing the ONT nanoporesat −180 mV even during a 1.5 h long experiment. In addition, itillustrates that this probe traverses the pores under applied voltage of−220 mV, and produces a high count of detectable events. (FIG. 5B) Fourconsecutive nanopore experiments conducted with the same but previouslyused flow cell that carries only around half, i.e., 250, workingnanopores. (i) buffer test at −180 mV, (ii) probe 122EXT at a load3-times less compared to regular load of 5 μM (corresponding to 0.38nmoles) tested at −180 mV (dashed line), (iii) no sample added, voltageraised to −220 mV and (iv) new sample with same load of probe 122EXT asin (ii), but prepared in 15% human serum-85% ONT buffer. (FIG. 5C) HPLCprofiles of (i) intact miRNA122, (ii) probe 2XdmiR122 at 0.5 μM, a10-fold lower concentration compared to the typical 5 μM sampleconcentration and (iii) mixture probe to target=1:2, also at a 0.5 μMconcentration in probe (hybrid). All three samples in >90% ONT bufferand monitored at 260 nm using HPLC method B (see Examples). The lattertwo samples were used, as is, for the nanopore experiments shown in FIG.5D. (FIG. 5D) Consecutive nanopore experiments on the same flow cell.(i) Buffer test, 1 h at −220 mV, (ii) probe 2XdmiR122, 3 h at −220 mVand (iii) hybrid sample with miRNA122, in excess over the probe, testedfor 1 h at −220 mV. This experiment confirms sensitivity in thedetection of 2XdmiR122, albeit via a 3 h long experiment, and confirmsthat hybridization with target results in severely reduced counts, i.e.,silencing. Data acquisition and analysis for the nanopore experiments asdescribed under FIG. 2A.

FIG. 6A,B. Targeting miRNA21 in a complex mixture. (FIG. 6A) HPLCprofiles of two samples analyzed with HPLC method B (see Examples): (i)probe dmiR21(OMe) at 0.15 nmole load and (ii) 1:2 mixture of this probewith miRNA21 at a 0.30 nmole probe load. The probe's HPLC profileexhibits two peaks, the larger one eluting after the minor one. Thisprofile is consistent with the determined value of 2.85 OsBp moietiesper molecule, on average, meaning that this preparation includesmolecules with 2 and molecules with 3 OsBp tags. The HPLC profile of the1:2 mixture of probe to target exhibits a single rather sharp peakeluting after a broad rather complex peak. It was confirmed that thesharp peak corresponds to the excess miRNA21 target. We attribute thebroad complex peak to multiple hybrids, the result of one target andmany probes, all complementary to the target, but each one of themcarrying OsBp moieties at a different nucleobase. It is reasonable toassume that the chromatography resolves these hybrids, as it resolvestopoisomers with such short osmylated oligos (Kanavarioti, A. (2016))(HPLC method B in Examples). The observation of distinct HPLC profilesbetween probe and mixture samples is consistent with hybridization.(FIG. 6B) Four nanopore MinION experiments two of them using the exactsamples analyzed by HPLC in (FIG. 6A): (i) probe dmiR21(OMe), tested for2 h at −180 mV, exhibited over 100,000 events, (ii) 1:2 mixture of thisprobe with miRNA21, tested for 1 h at −180 mV, exhibited negligiblecounts (hybrid). Two additional experiments tested the effect of excessnon-target RNA in the translocation properties of probe dmiR21(OMe) andits hybrid with miRNA21. The excess non-target RNA was at a 10-foldhigher load compared to the probe and was composed of equimolar amountof miRNA140 and a 100 nt long RNA. Nanopore experiments in the presenceof excess non-target RNA (iii) hybrid with miRNA21, 1 h at −180 mV, and(iv) probe dmiR21(OMe), 2 h at −200 mV. Higher voltage used here tocompensate for the hours that this flow cell had worked already, per ONTprotocol. The effect of the excess non-target RNA on the hybrid, if any,is not detectable, as the counts are too few. The effect of the excessnon-target RNA on the probe appears to be a profile shift and reducedcounts by a factor of 2, even though this reduction can be attributed tothe number of working nanopores that is also reduced by a factor ofabout 2. It was noted that probe dmiR21(OMe) translocates efficiently atan applied voltage of −180 mV, as it does not contain any adjacentosmylated dTs. Data acquisition and analysis as described under FIG. 2A.

FIG. 7A-D. Nanopore experiments targeting miRNA140 or miRNA21 atsingle-digit attomole load. (FIG. 7A) Consecutive nanopore experimentsconducted with the same flow cell: (i) buffer test, 1 h at −210 mV, (ii)probe 140EXT(mU) at a 47 fmole load, 1.5 h at −210 mV. (FIG. 7B)Consecutive nanopore experiments conducted with the same used flow cellwith less than 200 working nanopores: (i) buffer test, 1 h at −210 mV,(ii) probe 140EXT(mU) at a 3.5 amole load, 1.5 h at −210 mV and (iii)equimolar mixture of 140EXT(mU) and miRNA140, at 3.5 amole load each,1.5 h at −210 mV (hybrid). (FIG. 7C) HPLC profile of a sample withequimolar concentration of probe 21EXT(mU) and target miRNA21 using HPLCmethod B (Examples). The appearance of a single peak is consistent withhybridizations. Sample monitored at two different wavelengths to showthe absorbance at 312 nm attributed to the presence of the osmylatedprobe. (FIG. 7D) Consecutive nanopore experiments with the same flowcell; samples were first tested by HPLC, then diluted with ONT buffer bya factor of 10⁹ or 3×10⁸ for probe and hybrid, respectively (seeExamples). (i) buffer test, (ii) probe 21EX(mU) at 0.9 amole, and (iii)1:1 mixture of probe 21EXT(mU) with miRNA21 at 2.8 amole load each,(hybrid). Hybrid exhibits dramatically fewer counts compared to theprobe. Data acquisition and analysis for the nanopore experiments asdescribed under FIG. 2A.

FIG. 8. Samples of tsv files, obtained by running the OsBp_detectsoftware on fast-5 files. Left, sample probe T8(RNA); right, sample is amixture of d(CT)₁₀:T8(RNA)=1:1, both in about 90% ONT buffer (see FIG.2A for experimental conditions).

FIG. 9A-D. I-t recordings from two nanopore experiments ranging from 15to 60 s. Top, probe T8(RNA), i-t recordings from two different channels(see FIG. 9A,B). Bottom, mixture of d(CT)₁₀: T8(RNA)=1:1, i-t recordingsfrom the same two channels as on the top (see FIG. 9C,D). Vertical linesthat cross the x-axis (=0 pA) are instrument generated lines by voltagereversal, and not events. Top recordings show multiple and deep events,bottom recordings show few and shallow events. Shallow events areattributed to molecules bumping at the pore aperture, without traversingthe pore and they are not counted when selecting “All I_(r)/I_(o)<0.6”(see FIG. 8 and Examples).

FIG. 10A-D. HPLC profiles of individual components and their 1:1mixtures in a sample solvent about 90% ONT buffer. All HPLC profilesshown at 260 nm, with the exception of the osmylated probe and hybrid inthe top right profile that is shown at 272 nm and 312 nm. Hybridizationis evident by a mixture HPLC profile where the main peak elutes afterthe individual components. (FIG. 10A) Hybridization shown for two intactoligos, BJ1 and its complementary intact oligo (complement primerM13for(−20)). See FIG. 3B for comparable result with another pair of intactoligos. (FIG. 10B) Hybridization shown for probe BJ1 with 5 OsBp out of30 nt, and its complementary intact oligo (complement primerM13for(−20)). (FIG. 10C) Only partial hybridization is shown for intact BJ1and its complementary osmylated complement primerM13 for(−20) with 11OsBp out of 35 nt. (FIG. 10D) Hybridization is shown for intact BJ2 andits complementary osmylated complement primerM13 for(−41) with 6 OsBpout of 35 nt. HPLC method B is used for analysis (see Examples).

FIG. 11. Nanopore experiments with probes BJ2 and BJ4 show few events at−180 mV and numerous events at −220 mV. Probes BJ2 and BJ4 tested at−180 mV (dashed traces) show negligible number of counts. No new samplewas added, the voltage was raised to −220 mV and probes were tested at−220 mV (solid traces) and numerous events were detected. Both probesamples were used at a 0.2 nmole load. Experiments were conducted on thesame flow cell in the order BJ2 at −180 mV, BJ2 at −220 mV, BJ4 at −180mV and BJ4 at −220 mV; the duration of each experiment was 1 h. Dataacquisition and analysis as described under FIG. 2A. The dramaticdifference in counts at the different applied voltage clearly suggeststhat these probes and other probes, of similar design, do not traversethe proprietary CsGg nanopore at the lower voltage and require highapplied voltage of about −220 mV to translocate.

FIG. 12A,B. HPLC profiles of 1:1 mixtures (non-hybrids) of miRNA21 (FIG.12A) or miRNA21-A₁₅ (FIG. 12B) using probe 21EXT (with 8 T(OsBp), seesequence in Table 1). Samples in about 90% ONT buffer as the samplesolvent. HPLC profiles obtained with HPLC method B (Examples). HPLCprofile of the mixture sample matches closely the sum of the HPLCprofiles of the two components, providing evidence for no detectablehybridization in these two cases. The difference in these two cases isthat, due to the added A₁₅-tail, miRNA21-A₁₅ elutes couple of min latercompared to miRNA21.

FIG. 13. Effect of applied voltage on the counts observed with a mixtureof intact miRNA122 and miRNA140 at 10 μM each at −180 mV, at −200 mV, at−220 mV. Data shown at −180 mV are the same data as in FIG. 2B (Flongle)but normalized by multiplying with 10, as the MinION has about 10 timesmore working channels compared to the Flongle. Increased applied voltagereduces slightly the count of events, consistent with fastertranslocation and reduced detectability. An experiment with 10 μM ofmiRNA21-A₁₅ at −220 mV exhibits comparable counts with the combo ofmiRNA122 and miRNA140, but a distinct profile compared to the miRNA withno A₁₅-tail. The effect of voltage on the intact RNAs is in starkcontrast to the effect of voltage on most of the probes tested in thisstudy. No detectable difference in counts is observed with thecontrol/buffer between −200 and −220 mV.

FIG. 14. A good linear correlation was obtained for the number ofosmylated pyrimidines in an oligo, which is missing Ts, as a function ofthe number of U in a sequence. The correlation does not appear to dependheavily on whether or not the sequence is a DNA, RNA or carries 2′-OMegroups on all or on a portion of the bases (See Table 1). The linearcorrelation may be used to estimate the number of osmylated pyrimidinesper protocol c for any given sequence. The linear correlation isattributed to the observation that deoxyuridine (dU) is osmylated4.7-times faster compared to deoxycytidine (dC) (Ding, Y. & Kanavarioti,A. (2016)), and to the conditions of protocol c that yield only a smallpercentage of osmylated pyrimidines, and not a practically 100%osmylated oligo.

FIG. 15A-D. HPLC profiles of the four intact M13 primers and theircorresponding T-osmylated derivatives, using HPLC method A (seeExamples). Analysis of oligos in water as the sample solvent.T-osmylation of these oligos was conducted using protocol o (seeExamples). The reason osmylated oligos appear as multiple peaks isbecause top or bottom addition of OsBp to the C5-C6 double bond leads totopoisomers, that this chromatography resolves.

FIG. 16A-D. HPLC profiles of the four intact BJ1-4 and theircorresponding T-osmylated derivatives using HPLC method A. Analysis ofoligos in water as the sample solvent. T-osmylation of these oligos wasconducted using protocol o (see Examples).

FIG. 17A,B. HPLC profiles of the 2 intact complements of primerM13for(−20) (FIG. 17A) and primerM13 for(−41) (FIG. 17B) shown at 260 nm,and their corresponding T-osmylated derivatives shown at 272 nm and 312nm using HPLC method B (see Examples). Analysis of oligos in water asthe sample solvent. Briefly HPLC Method B is using the DNA PacPA200 HPLCcolumn from ThermoFisher Scientific at the 2×250 mm configuration with0.45 mL/min flow and 15° C. column compartment. Solvents are aqueous pH8.0±0.2 mobile phases A (MPA) and mobile phase B (MPB) with 25 mMTRIS.HCL buffer; MPB is 1.5 M NaCl. Initial conditions are 90% MPA-10%MPB, and the gradient is from 10% to 50% MPB in 20 min. The totalanalysis time including column equilibration is 30 min. T-osmylation ofthese oligos was conducted using protocol o (see Experimental Section).Right profile shows an atypical, but confirmed result, namely anosmylated conjugate that elutes later compared to the parent intactnucleic acid.

FIG. 18A-D. HPLC profiles of BJ2 TA(OMe) (FIG. 18A) and BJ2 AT(OMe)(FIG. 18B), as well as BJ1EXT(mU) (FIG. 18C) and BJ2EXT(mU) (FIG. 18D)shown at 260 nm and their corresponding T-osmylated derivatives shown at272 nm and 312 nm (sequences in Table 1). HPLC profiles obtained withHPLC method B (see FIG. 17A,B and Examples). Materials in water assample solvent. A nanopore experiment conducted with probe BJ2 TA(OMe)indicated excellent translocation properties with numerous counts at arelatively low probe load (FIG. 4D).

FIG. 19A-D. HPLC profiles of the intact miRNA21, miRNA122, miRNA140 andmiRNA21-A₁₅ (these are the −5p sequences) shown at 260 nm and theircorresponding partially osmylated derivatives; different materials areanalyzed at different sample load. Osmylation protocol o was used formiRNA21-A₁₅ and protocol c for the other 3 miRNAs (see Table 1 andExamples). HPLC method A used for the analysis of miRNA21-A₁₅ and HPLCmethod B for analysis of the other 3 miRNAs (see Examples). Materials inwater as the sample solvent.

FIG. 20A,B. (FIG. 20A) HPLC profiles of intact dmiR21 at 260 nm and theT-osmylation product at 272 nm and 312 nm. (FIG. 20B) HPLC profiles ofintact 21EXT and its T-osmylation product. Osmylation was carried outusing protocol o 40 min with 2.63 mM OsBp, earlier process where bipywas dissolved after adding OsO₄ (see Examples). Materials in water foranalysis, and analysis was done using HPLC method B (see Examples).

FIG. 21A-C. (FIG. 21A) HPLC profiles of the osmylation products ofdmiR21(OMe) using protocol b 30 min with 2.63 mM OsBp and protocol c 30min with 3.94 mM OsBp. A third protocol (d) with 30 min incubation using5.25 mM OsBp was used to osmylate this probe for the nanopore experiment(FIG. 6B). This was necessary because dmiR21(OMe) contains no Ts, andusing protocols b and c results in very low levels of osmylation anddiminished detectability. (FIG. 21B) probe 21EXT(mU) intact and itsosmylation products with the two different protocols; prep1, usingprotocol a 45 min with 2.63 mM OsBp and prep2 protocol b 30 min with2.63 mM OsBp. (FIG. 21C) repeat of FIG. 7C in order to compare the HPLCprofile of the hybrid with the HPLC profile of the probe alone (B,above) and see that they are distinct. Analysis was done using HPLCmethod B (see Examples).

FIG. 22A-D. HPLC profiles of the intact miRNA122 probes shown at 260 nmand their corresponding T-osmylated derivatives (the actual probes) at272 and 312 nm. Osmylation protocol o was used to osmylate dmiR122,2XdmiR122 and 122EXT. dmiR122(OMe) does not have any T, and wasosmylated using protocols b or c (see Table 1 for sequences and forprotocols). Materials were in water for analysis and analysis was doneusing HPLC method B (see Examples).

FIG. 23A-C. HPLC profiles of the intact miRNA140 probes shown at 260 nmand their corresponding osmylated derivatives (the actual probes) shownat 272 nm and 312 nm. HPLC profiles with dmiR140 (FIG. 23A) and2XdmiR140 (FIG. 23B) using osmylation protocol o, 40 min with 2.63 mMOsBp (earlier process, bipy not dissolved prior to OsO₄ addition. (FIG.23C) Probe 140EXT(mU) intact and its osmylation products with the twodifferent protocols; prep2, using protocol a, 45 min with 2.63 mM OsBp,and prep1 protocol b, 30 min with 2.63 mM OsBp. Materials in water foranalysis, and analysis was done using HPLC method B (see Examples).

FIG. 24A,B. (FIG. 24A) HPLC profiles of partially osmylated 100 nt RNAand 100 nt RNA(OMe) using osmylation protocol c (see Table 1 andExamples). (FIG. 24B) HPLC profiles of T-osmylated d(CT)₁₀ usingprotocol b. Materials in water as the sample solvent. HPLC method B wasused for all the samples and HPLC profiles are shown at 272 nm and 312nm. Osmylation protocols and HPLC method can be found in the Examples.

FIG. 25A,B. HPLC profiles of partially osmylated 22 nt RNAs. (FIG. 25A)complementary miRNA21 and (FIG. 25B), complementary miRNA122. Osmylationprotocol c was used, 30 min with 3.94 mM OsBp.

FIG. 26A,B. (FIG. 26A) Repeat of FIG. 6A in order to compare directlywith HPLC profiles to the right. (FIG. 26B) HPLC profiles of threesamples in 15% serum-85% ONT buffer: miRNA140 2 min incubation beforeanalysis, 100 nt RNA 30 min incubation before analysis. The longerincubation is why the degradation of 100 nt RNA appears more severecompared to the degradation of miRNA140. Mixture of dmiR21(OMe)(OsBp):miRNA21=1:2 in about 5% water-95% ONT buffer (FIG. 26A) and the samemixture in 15% serum-85% ONT buffer (FIG. 26B). The HPLC profiles appearcomparable suggesting that the hybrid suffers insignificant degradationin 15% serum-85% ONT buffer. HPLC method B used for these analyses (seeExamples).

DETAILED DESCRIPTION

The terms defined immediately below are more fully defined by referenceto the specification in its entirety. To the extent necessary to providedescriptive support, the subject matter and/or text of the appendedclaims is incorporated herein by reference in their entirety.

Definitions

It will be understood by all readers of this written description thatthe exemplary aspects and embodiments described and claimed herein canbe suitably practiced in the absence of any recited feature, element orstep that is, or is not, specifically disclosed herein.

The term “a” or “an” entity refers to one or more of that entity; forexample, “a probe,” is understood to represent one or more “probes.” Assuch, the terms “a” (or “an”), “one or more,” and “at least one” can beused interchangeably herein.

The term “and/or” where used herein is to be taken as specificdisclosure of each of the specified features or components with orwithout the other. Thus, “and/or” as used in a phrase such as “A and/orB” herein is intended to include “A and B,” “A or B,” “A” (alone), and“B” (alone). Likewise, “and/or” as used in a phrase such as “A, B,and/or C” is intended to encompass each of the following embodiments: A,B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C;A (alone); B (alone); and C (alone).

It is understood that wherever aspects are described herein with thelanguage “comprising,” otherwise analogous aspects described in terms of“consisting of” and/or “consisting essentially of” are also provided.

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure is related. For example, unlessotherwise specified, “complementary” base pairs refer to A/T, A/U, andG/C base pairing.

Numeric ranges are inclusive of the numbers defining the range. Evenwhen not explicitly identified by “and any range in between,” or thelike, where a list of values is recited, i.e., 1, 2, 3, or 4, thedisclosure specifically includes any range in between the values, i.e.,1 to 3, 1 to 4, 2 to 4, etc.

The headings provided herein are solely for ease of reference and arenot limitations of the various aspects or aspects of the disclosure,which can be had by reference to the specification as a whole.

As used herein, the term “identity,” i.e., “percent identity” to anamino acid sequence or to a nucleotide sequence disclosed herein refersto a relationship between two or more amino acid sequences or betweentwo or more nucleotide sequences. When a position in one sequence isoccupied by the same nucleic acid base or amino acid in thecorresponding position of the comparator sequence, the sequences aresaid to be “identical” at that position. The percentage “sequenceidentity” is calculated by determining the number of positions at whichthe identical nucleic acid base or amino acid occurs in both sequencesto yield the number of “identical” positions. The number of “identical”positions is then divided by the total number of positions in thecomparison window and multiplied by 100 to yield the percentage of“sequence identity.” Percentage of “sequence identity” is determined bycomparing two optimally aligned sequences over a comparison window. Inorder to optimally align sequences for comparison, the portion of anucleotide or amino acid sequence in the comparison window can compriseadditions or deletions termed gaps while the reference sequence is keptconstant. An optimal alignment is that alignment which, even with gaps,produces the greatest possible number of “identical” positions betweenthe reference and comparator sequences. Percentage “sequence identity”between two sequences can be determined using, i.e., the program “BLAST”which is available from the National Center for BiotechnologyInformation, and which program incorporates the programs BLASTN (fornucleotide sequence comparison) and BLASTP (for amino acid sequencecomparison), which programs are based on the algorithm of Karlin andAltschul (Proc. Natl. Acad. Sci. USA 90(12):5873-5877, 1993).

As used herein, the term “complementary” when referring to nucleic acidmolecules is given its standard definition for complementaryWatson-Crick base pairing as understood in the art.

The term “nucleic acid” is a well-known term of art and is used hereinto include DNA and RNA. Unless otherwise specified, a “nucleic acid”molecule and “polynucleotide” can be used interchangeably. A nucleicacid can comprise a conventional phosphodiester bond or anon-conventional bond (i.e., an amide bond, such as found in peptidenucleic acids (PNA)). By “isolated” nucleic acid it is intended anucleic acid molecule that has been removed from its native environment,such as a sample of genomic DNA obtained from a subject. Isolatedpolynucleotides or nucleic acids further include such molecules producedsynthetically.

As used herein, the terms “intact” or “native” when referring to anoligonucleotide means that the oligonucleotide is not osmylated.

As used herein, a “biological sample” is one derived from a subject suchas a human, animal, plant, bacteria, virus, fungus, or other type ofmulti-cellular or single-cellular life form. In certain aspects, thebiological sample can be obtained directly from the subject, such as bydrawing blood, collecting a urine sample, or a tissue or liquid biopsy.In certain aspects, the biological sample can be obtained indirectly,such as from biological evidence collected at a crime scene. In certainaspects, the biological sample is a bodily fluid such as blood, plasma,lymph, saliva, urine, amniotic fluid, spinal fluid, etc. In certainaspects, a biological sample is a fluid sample with components derivedfrom a tissue or cells suspended, dissolved, in solution, reconstitutedin, or the like, in the fluid sample. A “complex mixture” means a samplethat comprises various components such as nucleic acids, proteins,carbohydrates, etc. and/or varied nucleic acid molecules.

As used herein, an “event” or “count” is detected by applying a voltageacross the two compartments of a nanopore device, leading to a constantflow of electrolyte ions (I_(o)) via the pore, which is recorded as afunction of time (i-t). The passage of a single molecule through thepore reduces I_(o) to a lower level of residual ion current (I_(r)).This is recorded as an “event” with (I_(r)) and residence time (τ) (FIG.1B).

It has been discovered that a portable nanopore device from OxfordNanopore Technologies (ONT) can be repurposed to detect a DNA/RNApolynucleotide (target) in a complex mixture by conductingvoltage-driven ion-channel measurements. The detection and quantitationof the target was enabled by the use of a unique complementary probe ofthe present disclosure. Using a validated labeling technology, probesare tagged with a bulky Osmium tag (Osmium tetroxide 2,2′-bipyridine),in a way that preserves strong hybridization between probe and target.Untagged oligos traverse the nanopore relatively quickly compared to thedevice's acquisition rate and exhibit count of events comparable to thebaseline. Counts can be reported, for example, by a publicly availablesoftware, OsBp_detect (Kanavarioti, A., & Kang, A. See RNA(OsBp) eventdetection Python package in a public repository: hypertext transferprotocol secure github.com/kangaroo96/osbp_detect and for step-by-stepinstallation instructions see here: hypertext transfer protocol securegithub.com/kangaroo96/osbp_detect/blob/master/instructions.md). Due tothe presence of the bulky Osmium tag, osmium-tagged probes traverse moreslowly, producing multiple counts over the baseline, and can even bedetected in the single digit attomole (amole) range. In the presence ofthe target, however, the probe is “silenced”. Silencing is attributed toa double-stranded complex that for practical purposes of this disclosureare considered not to traverse the nanopore under the appliedconditions. Thus, the disclosed ready-to-use platform can be tailored asa diagnostic test to meet the requirements, for example, ofpoint-of-care circulating tumor DNA (ctDNA), cell free DNA (cfDNA)fragmented RNA, and microRNA (miRNA) detection and quantitation in bodyfluids.

Aspects of the present disclosure exploit selective labeling (alsoreferred to herein as tagging) of nucleic acids in an effort to enhancebase-to-base discrimination (Ding, Y. & Kanavarioti, A. (2016); SultanM., Kanavarioti, A. (2019); Kanavarioti, A. (2015)), utilizing Osmiumtetroxide 2,2′-bipyridine (OsBp) as the label/tag. OsBp is not reactivetowards purines and does not cleave the phosphodiester bond in DNA orRNA. OsBp adds to the C5-C6 double bond of the pyrimidines and forms twostrong C—O bonds without cleaving the pyrimidine ring (FIG. 1C) (Chang,C. H., Beer, M. & Marzilli, L. G. (1977); Palecek E. (1992); Reske T.,Surkus, A-E., Duwensee, H. & Flechsig G.-U. (2009); Kanavarioti, A. etal. (2012); Kanavarioti, A. (2016); Debnath, T. K. & Okamoto, A.(2018)). Reactivity of OsBp towards thymidine (T) is 28- and 7.5-timeshigher compared to the reactivity towards deoxycytidine (dC) anddeoxyuridine (dU), respectively (Ding, Y. & Kanavarioti, A. (2016)).Labeling condition protocols have been developed to selectively label Tin the presence of the other pyrimidines (Kanavarioti, A. et al.(2012)). Further, the inventor developed capillary electrophoresis (CE)and High-performance Liquid chromatography (HPLC) methods to measure theextent of labeling in short and long DNA and RNA (Kanavarioti, A. et al.(2012); Kanavarioti, A. (2016); see Examples). Voltage-drivenion-channel measurements were conducted using SiN solid-state nanopores(Henley, R. Y., Vazquez-Pagan, A. G., Johnson, M., Kanavarioti, A. &Wanunu, M. (2015)), the α-Hemolysin nanopore (Ding, Y. & Kanavarioti, A.(2016)), as well as the CsGg nanopore in the MinION (Sultan M.,Kanavarioti, A. (2019)), and demonstrated that all three platforms allowthe translocation of osmylated nucleic acids, and clearly discriminatethem from native nucleic acids. The discrimination is manifested as anevent with markedly lower I_(r) and longer τ and can be increased, forexample, by increasing the number and/or position of OsBp moietieslabeling an oligonucleotide. These features were utilized in order tosingle-out, detect, and count OsBp-tagged oligos in a complex mixture ofnative DNA and RNA.

FIG. 1D illustrates the concept behind nanopore-based identification andquantification of a target oligo in a complex mixture. Certain aspectsof this disclosure are enabled by a custom-designed OsBp-tagged oligo(probe) as described in detail elsewhere herein, that is at leastpartially complementary to a nucleic acid target molecule sufficient tocreate a hybridized double-stranded complex. The use of a complementaryoligo as a probe has been validated in several experimental nanoporeplatforms (Wanunu M, Dadosh T, Ray V, Jin J, McReynolds L, Drndić M.(2010); Xi, D. et al. (2016); Zahid, 0. K., Wang, F., Ruzicka, J. A.,Taylor, E. W. & Hall, A. R. (2016); Tian, K., Shi, R., Gu, A., Pennella,M., & Gu, L. Q. (2017); Hao, W., Haoran T., Cheng Y., & Yongxin, L.(2019)). These platforms however are complicated by the fact that theprobe is conjugated to a protein (Wanunu M, Dadosh T, Ray V, Jin J,McReynolds L, Drndić M. (2010)), a nanoparticle (Hao, W., Haoran T.,Cheng Y., & Yongxin, L. (2019)), a homopolymer (Xi, D. et al. (2016)),or a polypeptide (Tian, K., Shi, R., Gu, A., Pennella, M., & Gu, L. Q.(2017)). The detection in these platforms relies on counting the longblockades produced by the double-stranded hybrid complex bumping at thenanopore entry and practically “getting stuck”. None of these methodsreached commercial availability which hinders their broader use. Incontrast to earlier approaches based on detection of the hybrid (WanunuM, Dadosh T, Ray V, Jin J, McReynolds L, Drndić M. (2010); Xi, D. et al.(2016); Zahid, O. K., Wang, F., Ruzicka, J. A., Taylor, E. W. & Hall, A.R. (2016); Tian, K., Shi, R., Gu, A., Pennella, M., & Gu, L. Q. (2017);Hao, W., Haoran T., Cheng Y., & Yongxin, L. (2019)), aspects of thepresent disclosure detect the translocation of an osmylated probefacilitated by a relatively slow acquisition rate (for example, but notlimited by, MinION (3.012 kHz sampling rate, equivalent to reporting 3data points per 1 ms)) (Oxford Nanopore Technologies website:nanoporetech.com, under Resources/Publications). While the slow samplingrate misses many, most, or all translocation events of native DNA/RNAoligonucleotides, the events corresponding to the translocation of theOsBp-tagged probes are detected. In the absence of the nucleic acidtarget molecule (e.g., complementary probe binding partner), theosmylated probe traverses the nanopore and produces a detectable event.In the presence of the nucleic acid target molecule, the probe forms ahybridized complex with the target (e.g., a 1:1 double-stranded hybrid).The hybridized molecule does not fit through and does not traverse thenanopore (FIG. 1D). Hence the hybridized OsBp-tagged probe is“silenced”. In certain aspects, the hybridized complex, however, doesnot “clog” and prevent unhybridized single-stranded nucleic acids fromgoing through the pore. This can be achieved, for example, byincorporating automatic reversal of voltage to free the nanopores fromsuch unproductive, “clogging” occurrences. Therefore, to detect, testfor, and/or determine the presence or absence of a nucleic acid targetmolecule in a sample, an osmylated-probe is added to a particular samplewhich can be run on a nanopore platform or device by conductingvoltage-driven experiments. Absence of the nucleic acid target moleculein a sample can be, in certain aspects, construed from the detection ofnumerous events due to the tagged probe's translocation via thenanopore. The presence of the nucleic acid target molecule in a samplecan be, in certain aspects, construed from the absence of events, due tohybrid formation between the tagged probe and the target. Quantificationof the target can be based on the known concentration of the taggedprobe and 1:1 hybrid formation.

The presence of T(OsBp) moieties in the middle of a sequence is not afeature shared by many potential ctDNA, miRNA, or other such targets.Therefore, in certain aspects of probes of this disclosure, one, some,or all Ts in the sequence are replaced with uridine (U), deoxyuridine(dU), or 2′-OMe-Uridine (mU). Further, in certain aspects, one, some, orall the bases are modified as 2′-OMe. In certain aspects, one or moreadjacent T(OsBp) are added at the 3′-end or the 5′-end of the probeoligo. And, in some aspects, one or more additional dAs are added at the3′-end or the 5′-end of the probe oligo. Replacing Ts with U, dU, or mUreduces or eliminates the presence of OsBp within the complementarysequence which could hinder hybridization to the nucleic acid targetmolecule.

RNA/DNA hybrids are known to be more stable compared to DNA/DNA hybrids.Thus for example, in certain aspects, an RNA-based probe can target aDNA after addition of the probe to a biological sample to be tested. Incertain aspects, the DNA (e.g., a dsDNA) is relatively short, e.g., lessthan 100, 90, 80, 70, 60, 50, 40, 30, 28, 25, 24, 23, 22, 21, 20, 19, or18 nucleotides long. In certain aspects, the method includes adenaturation step before the RNA-based probe hybridizes to the nucleicacid target molecule, for example to denature a dsDNA target moleculeand/or remove any secondary structure of the probe. RNA probes exhibit adifferent nanopore profile compared to DNA-based probes, as indicated bycomparison of the (I_(r)/I_(o))_(max) in FIG. 2A with the corresponding(I_(r)/I_(o))_(max) in all the other figures representing probes. Thisfeature can lead to probe multiplexing in order to test more than onetarget at a time. Similarly, probes like dmiR21(OMe) that are missingthe 3 adjacent Ts were seen to translocate at −180 mV, while probes with3 adjacent Ts require −220 mV. Such distinctions, e.g., differences intranslocation between probes as a result of voltage, current, time,etc., and based on the quantity and/or location of osmylation of theprobe, can be exploited in order to multiplex probes. Distinct nanoporeprofiles will reveal which one of the probes is silenced. With just oneprobe, the test can conclusively identify the presence/absence of thetarget by comparing the total count of events of the probe alone to thetotal count of events to the mixture of the probe with the unknownsample. With a multiplexed test, for example, counts can be plotted ashistograms, in order to determine which probe is missing. Thus, certainaspects of this disclosure provide for multiplexing in order to test formore than one nucleic acid target molecule at a time, even within thesame test sample.

Certain aspects of this disclosure provide for ion-channel singlemolecule experiments conducted using, for example, portable,commercially available nanopore devices. Tested targets were DNA and RNAoligos and in certain aspects, exhibited a 9 orders of magnitude rangeof detection. This sensitivity approaches single-digit attomole targetsensitivity, for example from an 11 μL biological sample. Theseproperties enable detection and quantification of highly dilute samplessuch as ctDNA and miRNAs found in bodily fluids.

Provided for herein is a method for detecting the presence of a nucleicacid target molecule in a biological sample. One of ordinary skill inthe art will recognize that for any of the methods described below, ananalogous method can also be used to detect/verify the absence of anucleic acid target molecule in a biological sample if the criteria fordetecting the nucleic acid target sample are not met. In certainaspects, the method comprises contacting a test sample that comprises(i) a biological sample comprising a nucleic acid target molecule and(ii) an osmylated single-stranded oligonucleotide probe comprising atleast one pyrimidine residue covalently bonded to a substituted orunsubstituted Osmium tetroxide (OsO₄)-2,2′-bypyridine group (OsBpgroup). In certain aspects, the substitution occurs on the2,2′-bypyridine of the OsBp. In certain aspects, the test samplecomprises a sample buffer in which the biological sample is diluted. Thesample buffer composition can vary depending on the type of nanoporedevice/system, type of biological sample, etc., and can be determinedfor each circumstance. The biological sample is generally diluted sothat at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% of thevolume of the test sample is sample buffer with the remainder of thevolume being the biological sample and/or the solution comprising theprobe. One of ordinary skill in the art will recognize, however, thatthe smaller the volume of biological sample used, the smaller the amountof the nucleic acid target molecule that might be present, thusrequiring higher sensitivity. The osmylated pyrimidine can be athymidine (T), cytidine (C), deoxycytidine (dC), deoxyuridine (dU),uridine (U) or a derivative thereof. In certain aspects, at least oneosmylated pyrimidine residue is a thymidine residue (T). In certainaspects, the sequence of the probe is at least partially complementaryto the sequence of the nucleic acid target molecule, sufficient to allowthe formation of a hybridized probe/target complex. In certain aspects,at least a portion of the sequence of the probe is fully complementaryto at least a segment of the sequence of the nucleic acid targetmolecule. A nanopore device is used to detect in the test sample thenumber of events wherein unhybridized osmylated-probe traverses thenanopore. The number of events detected in the test sample can then becompared against some other value to determine or indirectly detect thepresence, or in some aspect the absence, of the nucleic acid targetmolecule in the test sample. In certain aspects, wherein the onlypotential source of the nucleic acid target molecule in the test sampleis that attributed to the biological sample, detection of the nucleicacid target molecule in test sample detects the nucleic acid targetmolecule in the biological sample. Unlike prior methods of detectingoligonucleotides on a nanopore system, the nanopore device/system ofthis disclosure does not require that the probe is conjugated to aprotein, a nanoparticle, a homopolymer, or a polypeptide for detectionof the probe. Thus, in certain aspects, the probe is not conjugated to aprotein, a nanoparticle, a homopolymer, or a polypeptide. Further,unlike prior nanopore detection methods, detection is not achieved bycounting long blockades produced by the hybridized probe/target complexblocking the nanopore or by melting of a hybridized probe/target complexin the nanopore.

(i) In certain aspect, the number of events detected in the test samplecan be compared to a “number of corresponding probe sample events.” Thenumber of corresponding probe sample events are those that are detectedin a probe sample or theoretically should be detected for a probesample, wherein unhybridized osmylated-probe traverses the nanopore inthe absence of the nucleic acid target. As explained in more detail andshown in representative Examples elsewhere herein, the presence of thenucleic acid target molecule in the target sample (such as coming fromthe biological sample or added as a control) leads to hybridization withthe complementary osmylated-probe, thus “silencing” the probe(preventing it from traversing the nanopore and resulting in adetectable event). Thus, a reduction in the number of events detected inthe test sample relative to the number of probe sample events isindicative of the formation of the hybridized probe/target complex andthus the presence of the nucleic acid target molecule in the testsample. Because unhybridized probe will traverse the nanopore and leadto detectable events, even in the presence of the nucleic acid targetmolecule if not all of the probe is hybridized, in certain aspects, theamount of probe should not greatly exceed or should not exceed theamount of nucleic acid target in the test sample. In certain aspects,the amount of probe should be about equal to or less than the amount ofnucleic acid target in the test sample. In certain aspects, the strengthof the hybridization between the target nucleic acid molecule and itscomplementary probe can also be taken into account. One of ordinaryskill in the art can determine based on available information androutine experimentation an approximate amount of nucleic acid targetmolecule in a particular type of sample and can further refine theamount of probe to use for optimal results. In certain aspects thereduction in the number of events is by at least a factor of two, by atleast a factor of three, or by at least a factor of four to confidentlydeduce that the observed reduction is due to the presence of the nucleicacid target molecule in the test sample and the formation of aprobe/target hybrid. This confidence level can vary, for example, fromprobe/target to probe/target, test conditions to test conditions, andnanopore device to nanopore device and can be determined by routineexperimentation of a given system. Thus, in certain aspects, a reductionin the number of events between those detected in a test sample and anumber of corresponding probe sample events is a reduction of at least afactor of two, at least a factor of three, or at least a factor of four

In certain aspects, the number of corresponding probe sample events isthe number of events contemporaneously detected in one or more probesamples at the time of detection of the number of events in the testsample. Contemporaneously detected means in the same general period oftime so that one of ordinary skill in the art would consider thedetection of events in the test sample and the detection of events inthe probe sample to be run as part of the same test or experiment, butnot necessarily occur in parallel. In certain aspects, contemporaneousdetection of events in the test sample and in the probe sample would usecommon reagents, such as from the same lot, to reduce variability. Incertain aspects, the reagents used for the contemporaneous detection areprovided together in a kit.

In certain aspects, the number of corresponding probe sample events is apredetermined value for a given amount of probe. A predetermined valueof the number of probe sample events for a given amount of probe can bedetermined empirically by running tests on probe samples with a knownamount of probe and detecting the number of events produced, forexample, on certain types of nanopore devices and under certainconditions, e.g., current, voltage, time, buffer conditions, age and/oramount of use of a nanopore device, etc. This predetermined value canthen be used for comparison purposes against the number of eventsdetected in various test samples under similar conditions. Thispredetermined value can be used for comparison to test samples with thesame or a similar amount of probe or the value can be extrapolated fordifferent amounts of probe. A predetermined value of the number ofcorresponding probe sample events for a given amount of probe can alsobe determined theoretically. A theoretical determination can be, but notneed be, informed by experimental observations.

In certain aspects, the number of osmylated probe sample events aredetected by a nanopore device in a probe sample and then the osmylatedprobe in the probe sample is combined with a biological sample to createa test sample. The number of events in this test sample can then bedetected by the nanopore device for comparison against the number ofosmylated probe sample events that were detected.

(ii) In certain aspects, the number of events detected in the testsample can be compared to the “noise” of a corresponding baseline samplethat does not contain any osmylated-probe. One of ordinary skill in theart would understand that even in the absence of osmylated-probe, andeven for just the sample buffer itself in the absence of any biologicalsample, the nanopore system will indicate a certain number of eventsreferred to herein as “noise.” One of ordinary skill in the art wouldalso understand that this noise can be accounted for in various ways(e.g., such as calibrating an instrument to zero out the noise) whichare non-limiting on the present methods. As explained in more detail andshown in representative Examples elsewhere herein, the presence of thenucleic acid target molecule in the target or control sample (such ascoming from the biological sample or added as a control) leads tohybridization with the complementary osmylated-probe, thus “silencing”the probe (preventing it from traversing the nanopore and resulting in adetectable event). Thus, an absence of an increase in the number ofevents detected in the test sample relative to the noise of thecorresponding baseline sample is indicative of the formation of thehybridized probe/target complex and thus the presence of the nucleicacid molecule in the test sample. Because unhybridized probe willtraverse the nanopore and lead to detectable events, even in thepresence of the nucleic acid target molecule if not all of the probe ishybridized, in certain aspects, the amount of probe should not greatlyexceed or should not exceed the amount of nucleic acid target in thetest sample. In certain aspects, the amount of probe should be aboutequal to or less than the amount of nucleic acid target in the testsample. In certain aspects, the strength of the hybridization betweenthe target nucleic acid molecule and its complementary probe can betaken into account. One of ordinary skill in the art can determine basedon available information and routine experimentation an approximateamount of nucleic acid target molecule in a particular type of sampleand can further refine the amount of probe to use for optimal results.In certain aspects, an absence of an increase in the number of events inthe test sample relative to the number of events in the baseline samplemeans an increase of less than a factor of two, less than a factor ofthree, or less than a factor of four to confidently deduce that theobserved reduction is due to the presence of the nucleic acid targetmolecule in the test sample and the formation of a probe/target hybrid.This confidence level can vary, for example, from probe/target toprobe/target, test conditions to test conditions, and nanopore device tonanopore device and can be determined by routine experimentation of agiven system. Thus, in certain aspects, an absence of an increase in thenumber of events in the test sample relative to the number of events inthe baseline sample means an increase of less than a factor of two, lessthan a factor of three, or less than a factor of four.

In certain aspects, the noise of a corresponding baseline sample iscontemporaneously determined in one or more baseline samples at the timeof detection of the number of events in the test sample.Contemporaneously detected means in the same general period of time sothat one of ordinary skill in the art would consider the detection ofevents in the test sample and the determination of noise in the baselinesample to occur as part of the same test or experiment, but notnecessarily occur in parallel. In certain aspects, contemporaneousdetection of events in the test sample and the determination of noise inthe baseline sample would use common reagents, such as from the samelot, to reduce variability. In certain aspects, the reagents used forthe contemporaneous detection are provided together in a kit.

In certain aspects, the noise of a corresponding baseline sample is apredetermined value. For example, in certain aspects, it may bepredetermined for a certain composition of a sample (e.g., type ofsample buffer, type of biological sample, concentration of biologicalsample in the test sample, etc.). A predetermined value of the noise ina baseline sample can be determined empirically by running tests onsamples with known compositions and determining the amount of noise, forexample, on certain types of nanopore devices and under certainconditions, e.g., current, voltage, time, buffer conditions, age ofnanopore device, etc. This predetermined value can then be used forcomparison purposes against the number of events detected in varioustest samples under similar conditions. This predetermined value can beused for comparison to test samples with the same or a similarcomposition or the value can be extrapolated for differing compositions,e.g., a higher or lower concentration of biological sample. Apredetermined value of the noise of a corresponding baseline sample of acertain composition can also be determined theoretically. A theoreticaldetermination can be, but not need be, informed by experimentalobservations.

In certain aspects, the noise of a corresponding baseline sample isdetermined for a nanopore device/system and then the osmylated probe isadded to the baseline sample, such as one comprising a biologicalsample, to create a test sample. The number of events in this testsample can then be detected by the nanopore device for comparisonagainst the amount of noise of the baseline sample.

(iii) In certain situations, it may be useful to use as a control asample comprising the nucleic acid target molecule and the complementaryosmylated probe, especially, for example, wherein the amount of nucleicacid target molecule in the control sample is known. Thus, in certainaspects, the number of events detected in the test sample can becompared to the number of corresponding control sample events whereinunhybridized osmylated-probe traverses the nanopore in the presence of aknown amount of the nucleic acid target molecule and/or known amount ofthe osmylated probe. Consistent with the use of a probe sample and testsample described above, a reduction in the number of events detected inthe test sample relative to the number of corresponding control sampleevents is indicative of the formation of the hybridized probe/targetcomplex and the presence of a higher amount of the nucleic acid targetmolecule in the test sample over the control sample. Use of such acontrol sample can be used to explore the hybridization between anucleic acid target molecule and its complementary probe and also totitrate and/or determine the optimum amount of probe to use for a givenamount of nucleic acid target molecule, even in the absence of thebiological sample. Such a use can be used to develop quantitativemethods of detecting the amount of nucleic acid target molecule in abiological sample.

As noted above, the 2,2′-bipyridine in OsBp, which is attached to apyrimidine in the oligonucleotide probe, can be substituted orunsubstituted. In certain aspects, it is substituted, for example,substituted with one or more methyl or ethyl groups.

In certain aspects, the nucleic acid target molecule can be a biomarkerand/or indicative of health, age, or a genotype associated with aphenotype or of a particular disease or disease state. In certainaspects, the nucleic acid target is a circulating tumor DNA (ctDNA),cell-free DNA (cfDNA), miRNA, a fragmented coding RNA, or a non-codingRNA. In certain aspects, the non-coding RNA is less than about 300 baseslong. In certain aspects, the nucleic acid target molecule is asingle-stranded nucleic acid molecule. In certain aspects, the nucleicacid target molecule is found in the biological sample as asingle-stranded nucleic acid molecule. In certain aspects, as found inthe biological sample, the nucleic acid target molecule is a strand of adouble-stranded nucleic acid molecule. Thus, in certain aspects, themethod comprises denaturing double-stranded nucleic acids and/or nucleicacids with secondary structure in the test sample, including the probe,to form single-stranded nucleic acid strands so that a single-strandedoligonucleotide probe can hybridize to a single-stranded targetmolecule.

As explained in detail elsewhere herein, in certain aspects, thenanopore device allows the voltage-driven translocation of osmylated andnon-osmylated single-stranded nucleic acids but prevents thetranslocation of double-stranded nucleic acids. While the methodsdisclosed herein can be performed using commercially available nanoporedevices, they are not limited to the type of nanopore device. In certainaspects, the nanopore device utilizes a nanopore having a minimal porediameter of about 1.3 nM to about 7.1 nM. In certain aspects, thenanopore device utilizes a Phi29, alpha-hemolysin, Aerolysin, MspA,CsGg, PA63, ClyA, FhuA, or SPP1 protein nanopore, or a bioengineeredderivative thereof.

As explained elsewhere herein, the voltage will vary depending onfactors such as the nanopore itself, the design of the osmylated probe,sample composition, age and/or amount of usage of the nanopore device,etc. Further, different voltages can be applied to specifically drivecertain nucleic acids through the nanopore but not others, such as todrive non-osmylated single-stranded nucleic acids across the pore beforeapplying the voltage needed to detect the osmylated probe or todifferentiate between osmylated probes of different designs such as in amultiplex test. In certain aspects, a voltage of about at least or atleast about −180 mV, −190 mV, −200 mV, −210 mV, −220 mV, −230 mV, −240mV, or −250 mV is applied to determine the presence of the target. Incertain aspects, a voltage of between any or any of about −180 mV, −190mV, −200 mV, −210 mV, −220 mV, −230 mV, or −240 mV and any or any ofabout −190 mV, −200 mV, −210 mV, −220 mV, −230 mV, or −240 mV, or −250mV is applied to determine the presence of the target. In certainaspects, a voltage of less than or less than about −200 mV, −190 mV,−180 mV, −170 mV, −160 mV, or −150 mV is applied before the voltageapplied to determine the presence of the target. In certain aspects, avoltage of between any or between any of about −200 mV, −190 mV, −180mV, −170 mV, or −160 mV and any or any of about 190 mV, −180 mV, −170mV, or −160 mV, −150 mV is applied before the voltage applied todetermine the presence of the target. It will be recognized by one ofordinary skill in the art that depending on the particular nanoporedevice and the characteristics of the incorporated nanopores, thisvoltage could be positive instead of negative, and it could be muchhigher in absolute terms.

Also, as explained in detail elsewhere herein, in certain aspects, themethod comprises using an algorithm to count events produced by thepassage of the probe, as reported by the time recording, to determine ifthe probe translocated freely via the nanopore. In certain aspects, thenanopore device allows for the distinction between different osmylatedprobes and multiplex detection of multiple different nucleic acidtargets in a test sample.

Using the methods and probes disclosed herein, in certain aspects, themethod can detect an amount of less than or less than about 1 pM, 100fM, 10 fM, 1 fM, 100 aM, 10 aM, 1 aM, or 0.1 aM of the nucleic acidtarget in the test sample. In certain aspects, the method can detect anamount of at least or at least about 0.1 aM, 1 aM, 10 aM, 100 aM, 1 fM,10 fM, 100 fM, or 1 pM of the nucleic acid target in the test sample.And, in certain aspects, the method can detect an amount of between anyor any of about 0.1 aM, 1 aM, 10 aM, 100 aM, 1 fM, 10 fM, or 100 fM andany or any of about 1 aM, 10 aM, 100 aM, 1 fM, 10 fM, 100 fM, of 1 pM ofthe nucleic acid target in the test sample.

In certain aspects, the method is quantitative for the amount of thenucleic acid target molecule in the test sample and/or biologicalsample.

Certain aspects of this disclosure are drawn to the design of probes foruse in detecting a nucleic acid target molecule in any of the methodsdescribed herein.

In certain aspects, the probe is DNA. In certain aspects, the DNAbackbone is modified. For example, in certain aspects, at least one ofthe sugars in the nucleic acid backbone are 2′-OMe-deoxyribose. Forexample 1, 2, 3, 4, 5 or more or 5%, 10%, 25%, 50%, 75%, 90%, 95% ormore or 100% of the sugars in the nucleic acid backbone are2′-OMe-deoxyribose.

In certain aspects, the probe is RNA. In certain aspects, the RNAbackbone is modified. For example, in certain aspects, at least one ofthe sugars in the nucleic acid backbone are 2′-OMe-ribose. For example1, 2, 3, 4, 5 or more or 5%, 10%, 25%, 50%, 75%, 90%, 95% or more or100% of the sugars in the nucleic acid backbone are 2′-OMe-ribose.

The length of the osmylated probe can be tailored to the length of thenucleic acid target molecule as well as for considerations such as easeand cost of synthesis, the amount of osmylation that would occur, andhybridization (e.g., a longer probe can have greater specificity andmore complementary base-pairing to a target than, e.g., a very shortprobe). In certain aspects, the osmylated probe has a length of aboutany of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,30, 35, 40, 50, 60, or 75 to any of about 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 30, 35, 40, 50, 60, 75, or 100. Incertain aspects, the osmylated probe has a length of about 12 to 50nucleotides. Because detection of the target nucleic acid molecule isachieved by forming a hybrid probe/target complex with the osmylatedprobe and because translocation of the probe through the nanopore is inits single-stranded form, in certain aspects it is preferable that theprobe not self-hybridize to itself, especially in the region ofcomplementarity with the target. Thus, in certain aspects, the portionof the probe that is at least partially complementary to the sequence ofthe nucleic acid target molecule lacks contiguous self-complementarysequences of more than 2 nucleotides. In certain aspects, the portion ofthe probe that is at least partially complementary to the sequence ofthe nucleic acid target molecule is unstructured. And in certainaspects, the portion of the probe that is at least partiallycomplementary to the sequence of the nucleic acid target molecule doesnot self-hybridize.

In certain aspects, an osmylated single-stranded oligonucleotide probemolecule comprises at least one pyrimidine residue covalently bonded toa substituted or unsubstituted Osmium tetroxide (0504)-2,2′-bypyridinegroup (OsBp group). In certain aspects, at least one osmylatedpyrimidine residue is a thymidine residue (T). It has been discoveredthat the amount of osmylation (the number of osmylated pyrimidines)influences how the probe transverses the nanopore and thus how it can bedetected as an event and how it can be distinguished from anon-osmylated single-stranded nucleic acid traversing the nanopore. Incertain embodiments, the osmylated probe comprises at least two, three,four, five, or six osmylated pyrimidine residues. In certainembodiments, the osmylated probe comprises two, three, four, five, orsix osmylated pyrimidine residues. As described elsewhere herein,certain methods preferably osmylate thymidine residues (T) over otherpyrimidines, thus allowing an even more nuanced approach to osmylatingthe oligonucleotide probe. In certain aspects, the osmylated probecomprises at least two, three, four, five, or six osmylated thymidineresidues (T). In certain aspects, the osmylated probe comprises two,three, four, five, or six osmylated thymidine residues (T). In additionto the amount/number of osmylated residues on the probe, the position ofthe osmylated residues in relation to each other can also influence howthe probe traverses the nanopore. The OsBp group on one residue canhinder the degrees of freedom of movement of an OsBp group on anadjacent residue, and thus how easily the probe can traverse thenanopore with the OsBp groups attached. Three adjacent residues furtherrestrict movement of the middle residue. Thus, in certain aspects, theosmylated probe comprises at least two, three, or four adjacentosmylated pyrimidine residues. In certain aspects, the osmylated probecomprises two, three, or four adjacent osmylated pyrimidine residues. Incertain aspects, the osmylated probe comprises at least two, three, orfour adjacent osmylated thymidine residues (T). In certain aspects, theosmylated probe comprises two, three, or four adjacent osmylatedthymidine residues (T). Further, the addition of deoxyadenosine (dA) oradenosine (A) residues to the end of the oligonucleotide probe may aidin traversal of the nanopore. In certain aspects, the osmylated probecomprises or comprise at least one, two, three, four, five, or sixadenosine residues (dA or A) at the 5′-end or 3′-end of the probe. Incertain aspects, the osmylated probe comprises or comprises at leastone, two, three, four, five, or six adenosine residues (dA or A) at the3′-end of the probe. In certain aspects, the osmylated probe comprisesor comprises at least one, two, three, four, five, or six adenosineresidues (dA or A) at the 5′-end of the probe. In certain aspects, oneor more of said 5′-end or 3′-end adenosine residues (dA or A) do nothybridize to the nucleic acid target molecule.

Osmylation of residues, especially within the portion of the probe thatis complementary or at least partially complementary to the nucleic acidtarget molecule can hinder hybridization with the nucleic acid targetmolecule. To avoid this and/or enhance hybridization, it may be usefulto locate osmylated pyrimidines, such as osmylated thymidine residues,in noncomplementary regions of the oligonucleotide probe sequence, suchas at the 5′-end or 3′-end and/or replacing thymidine residues in theregions of the probe complementary to the nucleic acid target with otherpyrimidines and taking advantage of the fact that under certain reactionconditions, thymidine residues can be preferentially osmylated overother pyrimidines.

In certain aspects, the osmylated probe does not comprise two or moreadjacent osmylated pyrimidine residues that are not located at the5′-end or 3′-end of the probe. And, in certain aspects, the osmylatedprobe does not comprise two or more adjacent osmylated thymidineresidues (T) that are not located at the 5′-end or 3′-end of the probe.In certain aspects, an osmylated probe comprises at least two, three, orfour adjacent osmylated pyrimidine residues located at the 5′- or 3′-endof the probe. In certain aspects, an osmylated probe comprises two,three, or four adjacent osmylated pyrimidine residues located at the 5′-or 3′-end of the probe. In certain aspects, the osmylated probecomprises at least two, three, or four adjacent osmylated thymidineresidues (T) located at the 5′-end or 3′-end of the probe. In certainaspects, the osmylated probe comprises two, three, or four adjacentosmylated thymidine residues (T) located at the 5′-end or 3′-end of theprobe. In certain aspects, one or more of said 5′-end or 3′-end adjacentpyrimidine residues does not hybridize to the nucleic acid targetmolecule. In certain aspects, none of said 5′-end or 3′-end adjacentpyrimidine residues hybridizes to the nucleic acid target molecule. Incertain of the forgoing aspects, the adjacent osmylatedpyrimidine/thymidine residues are located at the 5′-end of the probe. Incertain of the forgoing aspects, the adjacent osmylatedpyrimidine/thymidine residues are located at the 3′-end of the probe.

As disclosed herein, in certain aspects thymidine residues (T) can bepreferentially osmylated over other pyrimidines. Thus, in certainaspects, at least about 95%, 96%, 97%, 98%, 99%, or 100% of thethymidine residues (T) in the oligonucleotide probe molecule areosmylated and in certain aspects at least about 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, or 100% of pyrimidines present in the probe, other thanthymidine (T), are not osmylated. The DNA residue thymidine (T) and theRNA residue uridine (U) both are complementary to adenosine (A). Incertain aspects, the probe is DNA but at least one thymidine residue (T)in the probe sequence, other than adjacent thymidine residues (T)located at the 5′-end or 3′-end, is replaced by a uridine (U) or adeoxyuridine (dU) or a 2′-OMe-uridine (mU) residue. In certain aspects,at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of thethymidine (T) residues in the probe sequence, other than adjacentthymidine residues (T) located at the 5′-end or 3′-end, are replaced byuridine residues (U, dU, or mU). The result is an oligonucleotide probethat through the substitution of uridine (U) for thymidine (T), containsfewer or does not contain any osmylated thymidine residues (T) in theregion of the probe that hybridizes to the complementary nucleic acidtarget molecule but does contain adjacent osmylated thymidines at the3′-end or 5′-end of the probe.

There are numerous combinations of features that can be incorporatedinto the design of an osmylated oligonucleotide probe for use in themethods of this disclosure. In one representative design, the osmylatedprobe comprises or comprises at least two, three, or four adjacentosmylated thymidine residues (T) located at the 5′-end of the probe, theprobe is DNA but the thymidine residues (T) in the probe sequence, otherthan the adjacent thymidine residues (T) located at the 5′-end, arereplaced by a uridine residue (U, dU, or mU); and at least about 80%,85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of pyrimidines present in theprobe, other than thymidine (T), are not osmylated. Further, in certainaspects, at least one of the sugars in the nucleic acid backbone are2′-OMe-deoxyribose. In another representative design, the osmylatedprobe comprises three adjacent osmylated thymidine residues (T) locatedat the 5′-end of the probe, the probe is DNA but the thymidine residues(T) in the probe sequence, other than the three adjacent thymidineresidues (T) located at the 5′-end, are replaced by a uridine residue(U, dU, or mU); and all of the pyrimidines present in the probe, otherthan thymidine (T), are not osmylated. Further, in certain aspects, someor all of the sugars in the nucleic acid backbone are2′-OMe-deoxyribose.

As described in more detail elsewhere herein, in certain aspects, theosmylated probe can be prepared by reacting an aqueous solutioncomprising a substituted or unsubstituted 2,2′-bipyridine and an OsO₄(osmylating reagent) with an oligonucleotide to form the2,2′-bipyridine-OsO₄-conjugated probe. In certain aspects, theconjugated probe is purified from excess osmylating reagent. In certainaspects, the 2,2′-bipyridine/OsO₄ ratio in the solution is equimolar ornear equimolar. For example, in certain aspects, the2,2′-bipyridine/OsO₄ ratio in the solution is about 0.80/1.0, 0.85/1.0,0.90/1.0, 0.95/1.0, 0.97/1.0, 0.98/1.0, 0.99/1.0, 1.0/1.0, 1.0/0.99,1.0/0.98, 1.0/0.97, 1.0/0.95, 1.0/0.90, 1.0/0.85, or 1.0/0.80.Alternative methods of preparing the osmylated probe exist and arecontemplated. For example, in certain aspects, the osmylated probe isprepared by ligation of a (dT(OsBp))_(n) oligo to the 5′-end or 3′-endof the probe, wherein n is 2, 3, or 4.

Provided for herein are kits comprising reagents for performing themethods of this disclosure. In certain aspects, a kit comprises anosmylated probe of this disclosure and a control nucleic acid targetmolecule that can hybridize to the probe. In certain aspects, thecontrol nucleic acid target comprises a nucleic acid sequence of actDNA, cfDNA, miRNA, a fragmented coding RNA, or a non-coding RNA. Incertain aspects, the non-coding RNA is less than about 300 bases long.

Also provided for herein is the use of a probe of this disclosure forthe detection of a nucleic acid target molecule with a nanopore device,wherein the nucleic acid target molecule is optionally a ctDNA, cfDNA,miRNA, a fragmented coding RNA, or a non-coding RNA. In certain aspects,the non-coding RNA is less than about 300 bases long.

EXAMPLES

Materials and Methods

Oligos and other Reagents

Custom-made RNA oligos were purchased from Dharmacon (Horizon DiscoveryGroup). Custom-made deoxyoligos were purchased from Integrated DNATechnologies (IDT). Sequences and UV/Vis properties of their osmylatedderivatives are listed in Table 1. The purity of oligos was tested byHPLC and typically found to be >85%. Oligos were diluted with AmbionNuclease-free water, not DEPC treated, from Thermo Fisher Scientifictypically to 100 or 200 μM stock solutions and stored at −20° C. HPLCprofiles from the analyses of both the intact and the osmylated oligoare included elsewhere herein. Buffer DNase-free and RNAse-free TRIS.HCl1.0 M pH 8.0 Ultrapure was purchased from Invitrogen and used to preparethe HPLC mobile phase. NaCl crystalline ACS min 99.0% was obtained fromAlfa Aesar. Distilled water from Alhambra was used for preparation ofHPLC mobile phase. A 4% aqueous osmium tetroxide solution (0.1575 M OsO₄in ampules at 2 mL each) was purchased from Electron MicroscopySciences. 2,2′-Bipyridine 99+% (bipy) was purchased from Acros Organics.Human Serum from human male AB plasma and NaOH 1N Bioreagent werepurchased from Sigma. ss M13mp18, primer M13(fwd6097), NEBuffer 2.1, andKlenow Fragment of DNA Polymerase I (3′-5 exo)(M0212) were kindlyprovided by New England Biolabs, Ipswich, Mass., USA.

While not limiting on the probes of this disclosure, expense and productquality considerations led to selection of DNA over RNA oligos as probesfor most example experiments. These experiments revolved aroundoptimizing the probe's design to make it of general applicability, andaptly detectable by available nanopore systems (e.g., the ONT/CsGgnanopore). In certain aspects, a probe that successfully identifiedmiRNA targets at the single-digit attomole level has sequencecomplementary or at least partially complementary to the nucleic acidtarget molecule, 2′-OMeU (mU) replacing T within the sequence, 3additional adjacent T residues at the 5′-end, and 3 added dA residues atthe 3′-end. The oligo is osmylated to add 4 to 5 OsBp tags per molecule,3 of which occupy the 5′-end, and the other 1 or 2 are randomlyallocated within the sequence. Due to the heavy crowding at the one endof the probe, applied voltage in the range of −210±10 mV was requiredfor translocation and detection of this illustrative probe. This featurewas advantageous, as it allows for depletion of non-target material at−180 mV prior to performing the diagnostic experiment at −210 mV. Probesmay also be multiplexed when using probe designs that exhibit distinctnanopore profiles. Preliminary experiments in 15% human serum suggestthat probes and the resulting hybrids are stable in such medium, andshow the feasibility for identifying short DNA and RNA from body fluidsamples using the OsBp with a nanopore platform.

Example 1

(Pyrimidine)OsBp is a Chromophore

Selective labeling of a nucleic acid requires an assay for qualitycontrol. Addition of OsBp to the C5-C6 Py double bond and formation ofPy(OsBp) creates a new chromophore in the wavelength range of 300 to 320nm (Kanavarioti, A. et al. (2012)), where nucleic acids exhibitnegligible absorbance. This observation was exploited using a deoxyoligotraining set to show that extent of osmylation can be measured using theequation: R(312/272)=2×(No of osmylated pyrimidines/total nt ofnucleotides) (Kanavarioti, A. et al. (2012)). Value R(312/272) is theratio of the observed peak absorbance at 312 nm over the observed peakabsorbance at 272 nm (the peak shape could be sharp or broad or multiplepeaks) (Kanavarioti, A. (2016)). The wavelengths 312 nm and 272 nm werechosen in order to maximize the effect and to equalize contributions bydifferent pyrimidines (Kanavarioti, A. (2016)). Absorbance at 272 nm isabout 75% of the absorbance at 260 nm practically for either intact orosmylated nucleic acids. Using the ratio R instead of the absorbance at312 nm normalizes the measurement, and minimizes instrument samplingvariation.

As deduced by experimenting with an oligo training set, when observedvalue R(312/272)=2×(# of pyrimidines/total # of nucleotides), osmylationis practically 100% complete and all the pyrimidines carry one OsBpmoiety (Kanavarioti, A. et al. (2012); Kanavarioti, A. (2016)). Whenobserved R(312/272)<2×(# of pyrimidines/total # of nucleotides),osmylation is partial and the number of osmylated pyrimidines or OsBpmoieties can be calculated based on the same equation (see Table 1).With partial osmylation the number obtained from the equation refers toOsBp moieties, on average. Molecules carry an integer number of OsBpmoieties and therefore some molecules will have fewer, and somemolecules will have more than the calculated value distributed in astatistically unbiased manner. It has been shown that osmylation occursrandomly, but depends on the relative reactivity of OsBp for thepyrimidines. Relative reactivity towards osmylation, determined bykinetic measurements at 26° C. in water using deoxyribooligos T/dC=28,dU/dC=3.75 and therefore T/dU=7.5 (Ding, Y. & Kanavarioti, (2016)), andusing ribooligos U/C=4.7 and 5-MeU/C=44; hence 5-MeU/U=9.3, with 5-MeUcarrying the identical nucleobase as T, (Kanavarioti, A. (2016)).Because of the markedly higher reactivity of OsBp towards T, compared todU or U and dC or C, conditions can be found to osmylate practically100% all Ts, while some dU and very few dC become osmylated(Kanavarioti, A. et al. (2012)). This dramatically higher reactivity wasexploited by adding 3 Ts at the 5′-end of a probe, replacing the Ts inthe sequence with dU or mU, and optimizing the manufacturing process asdetailed below.

Example 2

Manufacturing of OsBp-Nucleic Acids

OsBp reagent was prepared by weighing the equivalent of 15.7 mM of bipy(49.2 mg) in a 20 mL scintillation vial, adding 18 mL of water andstirring at room temperature until bipy dissolved, followed bytransferring the full content of a 2 mL 4% OsO₄ solution supplied in anampule. Dissolving bipy in water before the addition of OsO₄ (protocolsa, b, c, and d) results in a more consistent and potent preparationcompared to dissolving bipy after the addition of OsO₄ (protocol o). Thetransfer was done using a glass pipette inside a safety hood (MSDS andInformation is obtained from the following link to UCLA ChemistyDepartment on the world wide web atchemistry.ucla.edu/sites/default/files/safety/sop/SOP_Osmium_Tetroxide.pdf).The resulting solution is an aqueous 20 mL 15.75 mM OsBp (0.4%) stocksolution, equimolar in OsO₄ and bipy. The concentration of the OsBpstock solution is limited by the solubility of bipy in water and addingOsO₄ does not increase it, as the complex has a low associationconstant. OsBp complex represents an approximate 5% of the total, asmeasured by CE (Kanavarioti, A. et al. (2012)). Care should be takenthat this preparation, as well as any other work using OsBp is conductedin stoppered glass vials in a well-ventilated area. Leftover solutionsof OsO₄ and/or OsBp may be mixed with corn oil to neutralize unreactedOsO₄ and properly discarded based on specific local regulations (MSDSand Information is obtained from the following link to UCLA ChemistyDepartment on the world wide web atchemistry.ucla.edu/sites/default/files/safety/sop/SOP_Osmium_Tetroxide.pdf).The freshly prepared OsBp stock solution was dispensed in HPLC vials andkept at −20° C. Each vial can be stored at 4° C. and used for a monthwithout loss of potency; typical pipette tips can be used formanufacturing of OsBp-labeled nucleic acids. OsBp stock solutions shouldbe validated before first use by running a known reaction. Forosmylation reactions a 20-fold excess of OsBp over the reactivepyrimidine in monomer equivalents was used to ensure pseudo-first orderkinetics, and to assure successful use of protocols. Manufacturingconditions, i.e., OsBp concentration and labeling duration variessignificantly depending on the presence of T, and the desired result ofosmylating all pyrimidines, T only, or just a fraction of dC and dU inDNA and a fraction of C and U in RNA. For the purpose of this study,when T-osmylation is required, protocol b is recommended, and whenpartial osmylation of U and C is required, protocol c or protocol d arerecommended. These choices were facilitated by testing additionalprotocols (o and a), as identified in Table 1. Quenching of theosmylation reaction occurs upon purification. Purification from excessOsBp was done with spin columns (TC-100 FC from TrimGen Corporation)according to the manufacturer's instructions. Briefly, spin columns arefilled with the manufacturer's proprietary solution and centrifuged at4,000 rpm for 4 min; the resulting solution and the microcentrifuge tubeare discarded. Then 40 to 1204, of an osmylation reaction mixture istransferred to the spin column and centrifuged at 4,000 rpm for 4 minusing a clean microcentrifuge tube. The centrifuged solution is thepurified osmylated oligo. This purification method retains thevolume/concentration of the sample, and close to 100% recovery of oligois achieved.

Recommended protocol b for thymidine (T)-osmylation is 30±2 minincubation with 2.6 mM OsBp in water at room temperature (see tableunder b). Recommended protocol for partial U- and C-osmylation of probesthat do not carry dTs is 30±2 min with 3.9 mM OsBp in water at roomtemperature (see Table 1 under c) or 30±2 min with 5.2 mM OsBp protocol(see Table 1 under d). There were two additional protocols tested, butthese were found to be less optimal: Protocol where bipy was notdissolved in water prior to the OsO₄ addition and used 40 min incubationwith 2.6 mM OsBp (see Table 1 under o) and the protocol using 45 minincubation with 2.6 mM OsBp (see Table 1 under a). The presence of2′-OMe groups does not affect markedly the extent of osmylation, as seenby comparable osmylation extent for 100 nt RNA and 100 nt RNA(2′-OMe)that carries about 50% 2′-OMe bases. The extent of osmylation can beaffected by the number of U in the oligo as seen in FIG. 14. This isbecause the reactivity of OsBp towards dU is 3.75 times higher comparedto the reactivity towards dC and the reactivity of OsBp towards U is 4.7times higher compared to the reactivity towards C. Because theosmylation protocols do not go to completion dU(OsBp) or mU(OsBp) arekinetically preferred over dC(OsBp) and U(OsBp) preferred over C(OsBp).With protocol b, osmylation of other pyrimidines is negligible comparedto T-osmylation, but with protocol c, the extent of osmylation towardsU, dU, mU and dC is measurable. FIG. 14 suggests a linear correlationbetween the number of OsBp moieties and the number of U present in anoligo spanning from 22 nt to 100 nt oligos. One may use the slope=0.43of the graph to estimate extent of osmylation with protocol c for anyoligo based on the number of Us in the sequence.

2 mL HPLC vials fitted with 120 μL glass inserts were used to carry themanufacturing reactions. Removing the reaction product from theseinserts and transferring it onto the purification spin column requires along and narrow 20 μL pipette tip. Osmylated nucleic acids are as stableas the corresponding nucleic acid, and the OsBp label is unreactive.They can be stored in the 1.5 mL microcentrifuge tubes, at −20° C. foryears. The concentrations of 2.6, 3.9 and 5.2 mM correspond to ⅙, ¼ and⅓ dilutions of the 15.75 mM OsBp stock solution, respectively.Deviations from these two protocols are included in the table,identified as protocols o and a. The additional protocol (d) was used inorder to achieve higher extent of osmylation with probes that do notcontain any T, such as dmiR21(OMe), and make it a detectable probe (seeBrief Description of FIG. 21A).

Example 3

Enzymatic Elongation Reactions

The ability of DNA polymerase to extend an osmylated primer was examinedin vitro using ssM13mp18 annealed to unmodified and osmylatedoligonucleotides. ssM13mp18 at a concentration of 42 nM was mixed with0.42 μM primer in NEBuffer 2.1 (New England Biolabs). Samples wereheated to 90° C. for 30 seconds and cooled to 25° C. at 0.1° C./sec.Polymerization reactions contained these annealed complexes (8.4 nMssM13mp18), 1.25×NEBuffer 2.1, 0.25 mM each of dGTP, dATP, and dTTP,0.025 mM α-[³²P] dCTP, and 7.7 U/ml Klenow Fragment of DNA Polymerase I(3′-5′ exo-) (NEB, M0212). Reactions were incubated at 37° C. andincorporation of labeled dCMP was monitored by an acid precipitationassay. Time points were taken at 5, 10, and 20 minutes.

As seen in FIG. 3A, no incorporation was noted when no oligonucleotidewas added, or if the oligonucleotide, primerM13rev(−48), was notcomplementary to the ssM13mp18. In contrast, most oligonucleotidespredicted to anneal to the DNA template gave robust incorporation, evenwhen osmylated, with maximal incorporation corresponding to roughly oneround of replication on the M13mp18 DNA template. Incorporation notedfor M13(fwd6097), BJ2, BJ3, and BJ4 were equivalent despite interiorsingle base mismatches within BJ3 and BJ4 (see sequences in Table 1).Despite overall equivalent osmylation levels for BJ2, BJ3 and BJ4,markedly lower levels of incorporation were noted for BJ1, most likelydue to presence of OsBp at the terminal 3′-T(OsBp) residue. Controlexperiments mixing BJ1 with M13(fwd6097) displayed full incorporation,discounting soluble inhibitors as the cause of low incorporation withBJ1 (data not shown).

Example 4

HPLC Methods

An HPLC method to assess oligo purity was developed and used here toassess purity of the oligos listed in the Table 1. This method wasoptimized and used to assess hybridization between two oligos in amixture; validation of this method was conducted using a 1:1 mixture ofintact oligos known to hybridize (see FIG. 3B and FIG. 10A). Analyseswere conducted automatically using a thermostatted autosampler. HPLCpeaks were detected and identified using a diode array detector (DAD) inthe UV-vis region 200-450 nm. The chromatograms were recorded at 260,272, and 312 nm and reported here selectively. Samples were preparedwith RNAse free water, but buffers were not.

For HPLC analysis an Agilent 1100/1200 LC HPLC equipped with a binarypump was used, Diode Array Detector (DAD), a 1290 InfinityAutosampler/Thermostat, and Chemstation software Rev.B.04.01 SP1 fordata acquisition and processing. For sample analyses IEX HPLC columnDNAPac PA200 from ThermoFisher Scientific (Dionex) was used in a 2×250mm configuration. The performance of the instrument and the column wasqualified using standards every time ahead as well as after analysis ofresearch samples. The HPLC method was developed to assess hybridizationin a sample approximately 90% in aqueous ONT buffer at pH 8 and columnthermostat at 35° C. This HPLC method (identified as HPLC method B) usesthe DNAPac PA200 column with a 0.45 mL/min flow, mobile phase A (MPA)aqueous 25 mM TRIS.HCl pH 8 buffer, mobile phase B (MPB) aqueous 1.5MNaCl in a 25 mM TRIS.HCl pH 8 buffer, a gradient from 10% MPB to 50% MPBin 20 min and an additional 10 min for wash and equilibration to initialconditions, i.e. 90% MPA. Column temperature was set at 35° C. to mimicthe flow cell temperature. To include the 100 nt RNA the chromatographywas modified to HPLC Method C. Specifically the gradient was madesteeper, 10% MPB to 75% MPB in 20 min, and everything else remained asis. Because of the approximately neutral pH the long 100 nt RNA elutesas a broad peak resembling a mixture. This is because aqueous pH 8 doesnot denature the various conformations of a long RNA, as reportedearlier (Kanavarioti, A. (2019)). ONT buffer has a UV-Vis component thatin this chromatography elutes in the void volume and does not interferewith the analysis of the samples. Sample injection volume was typicallyat 5 μL, and not higher than 10 μL. The hybridization test can be usedin conjunction with samples in ONT buffer or any other medium thatfavors complexation. Some of the oligos and their osmylated derivativeswere analyzed using a method identified as HPLC method A, which isrecommended for oligo purity analysis (Kanavarioti, A. (2019)), but notsuitable to test hybridization. HPLC method A uses the DNAPac PA200column with a 0.45 mL/min flow, and column temperature at 30° C. Mobilephase A (MPA) aqueous pH 12.0±0.2 with 0.01 N NaOH, mobile phase B (MPB)aqueous 1.5M NaCl in pH 12.0±0.2 with 0.01 N NaOH, a gradient from 20%MPB to 95% MPB in 12 min and an additional 8 min for wash andequilibration to initial conditions, i.e. 80% MPA.

Example 5

Single Molecule Ion-Channel Conductance Experiments with the CsGgNanopore in a MinION or a Flongle (ONT Platform)

ONT instructions were followed in order to remove air bubbles from theflow cell, flush the storage solution with ONT Flush buffer, add sample,or store the flow cell, as needed. Instructions for MinION (754, sample)and Flongle (304, sample) are obtained from the protocols found on theONT website. Flongle flow cells require an adaptor but work on the samedevice as the MinION flow cells. The software MINKNOW to run thenanopore experiments was downloaded on a MacBook Pro laptop used forthese experiments. All the functions necessary to test the flow cell andrun the experiments are done via the MINKNOW software tool. Raw datafiles were acquired in fast-5 format, which were then analyzed by theOsBp_detect software. Size of fast-5 files for the experiments depend onthe flow cell and experiment duration and vary between 1.5 and 6 GB.Fast-5 files can be directly visualized in MatLab (from Mathworks) 2Dformat, once the experiment is completed. MINKNOW allows for monitoringin real time any chosen channel, up to 10 channels, so one doesn't needto wait for the experiment to be done to see the i-t recordings.

Added samples were either untagged oligos, osmylated oligos, or mixturesthereof. Concentrations were typically at or below 10 μM oligo in noless than 80% of ONT buffer. No library was prepared, and no processingenzyme was added, such that all the translocations reported here areunassisted and voltage-driven. Experiments lasted no longer than 1.5 h,but the same experiment could be extended by stopping it, and restartingit later, or next day without adding a new sample. Running the same flowcell for more than 4 hours per day was avoided, and flow cells werestored temporarily in ONT buffer. Cleaning with buffer the flow cellbefore running a new sample was done right before the next experiment.In most cases the first experiment was a “buffer test” to assess theflow cell's baseline. Duration of the buffer test was kept as short aspossible, because the nanopores of a flow cell did not last for morethan 15 hours under our experimental conditions. Per ONT protocol,applied voltage was raised by about 10 mV for every 5 working hours, inan attempt to keep the open pore ionic current (I_(o)) constant. This iswhy experiments compared to one another are done at seemingly differentapplied voltage. The MinION flow cell has over 2000 nanopores, but only512 are monitored simultaneously. During the first few experiments on aflow cell, nanopores become inactive, but they are replaced with newworking ones. Therefore, the first 4 to 5 experiments are practicallydone with the same number of pores. After that the number of workingnanopores decreases by 5 to 10% per hour. It is worth noting that whilemost of the working nanopores exhibited comparable number of eventswithin a factor of 5 from lowest to highest, a small percentage deviatedand recorded markedly higher count of events. These pores were called“outliers” and it was estimated approximately 2.5% of nanopores to beoutliers. The analysis of the data presented here includes all channels.Additional analysis of all the experiments was performed by excludingthe outliers, and even though the actual counts were different, thetrends and conclusions are identical to the ones presented here.

Example 6

Event Detection Algorithm—OsBp_Detect

Let y be an ordered sequence of real values representing a typicaltime-series obtained from a single Nanopore. We categorize regions of yinto one of two states: current from an open channel, I_(o), where theNanopore is unoccupied, and the residual current when some translocationevent is taking place, I_(r). For the high-throughput characterizationof a single translocation event corresponding to an OsBp-taggedoligonucleotide, we propose a segmentation algorithm that can determinethe start and end positions in y for all events of interest based onuser-defined thresholds. The analysis pipeline is divided into threesteps:

1. Baseline current estimation

2. Identification of potential candidate events

3. Event filtering based on event features

First, the baseline I_(o), b_(o), is established by taking a median ofthe signal values between estimated lower and upper bounds,{o_(low),o_(up)}, of the open current:b_(o)=med({i:i∈y,o_(low)<i<o_(up)}).

While the signal noise is dependent on the nanopore platform used, thesharp transitions between the I_(o) and I_(r) states from osmium-taggedoligos permit the use of a single threshold-based parser for eventdetection pipeline. With this approach, events are identified if theypass a set threshold away from the local baseline level. The thresholdis defined by parameter ball which should be low enough to capture asmany translocation events as possible. By default,ball=b_(o)·(1−(10·σ_(o)/b_(o))) where σ_(o) represents the signal noiseof the open current. The noise constant, σ_(o), is determined bysplitting y into small segments (segment size used, n=100,000) andcalculating the global standard deviation of the open current signalvalues, σ({i:i∈y,o_(low)<i<o_(up)}). The σ_(o) value additionallyprovides a useful quality control metric to detect pores with unstablebaselines and large events rates or pores that have been blocked.

Finally, for the qualification of valid translocation events, twofiltering conditions were applied to the identified events. The filterscorrespond to the minimum and maximum event length defined byparameters, {t_(min),t_(tax)}, and the range of lowest residual current,{b_(min),b_(max)}, expressed as a ratio with respect to b₀. The eventlength thresholds, {t_(min),t_(max)}, can be adjusted to monitor thespeed of translocation whereas {b_(min),b_(max)} enables the separationof OsBp-tagged and untagged oligo species. Let τ₁ and τ₂ represent thestarting and ending indices for any given event in y. For y_(τ1:τ2) tobe classified as a potential OsBp translocation event, both of thefollowing conditions must hold:

$⪢ t_{\min} < {\tau_{2} - \tau_{1}} < t_{\max} ⪢ b_{\min} < \frac{\min\left( y_{\tau_{1}:\tau_{2}} \right)}{b_{o}} < b_{\max}$The event detection pipeline is available as a Python library,‘osbp_detect’. A cross-platform graphical user interface has beenincluded, to enable direct reporting of translocation events from ONTbulk fast5 files (hypertext transfer protocol securegithub.com/kangaroo96/osbp_detect).

TABLE 1 List of tested DNA or RNA oligos. The 3 miRNAs tested here havethe sequence of the corresponding miRNA-5p. Oligo ID, sequence, numberof thymidines over total nucleotides (T/total nt), number of pyrimidinesover total nucleotides (Py/nt), theoretical R(312/272) for T(OsBp) (seefootnote and Examples), Observed R(312/272) is the ratio of the observedareas under the HPLC peak at 312nm over the area at 272nm; HPLCanalytical profiles for each oligo and its osmylated conjugate areincluded elsewhere herein. The number of Py(OsBp) or OsBp moieties, onaverage, depends on the protocol used for osmylation and is calculatedas described in footnote 3 below. Sequence; sequences are alldeoxyribose, with the ID, exception of the three Theoretical DNA unlessmiRNAs, T8 and 100nt RNA; T/ R(312/272)¹ Observed Py/ No of OsBp³ SEQID/. identified as RNA (mU is 2′-OMeU) total nt T(OsBp) only R(312/272)²total nt on average NO: PrimerM13for(-20) GTA AAA CGA CGG CCA 2/17 0.2350.230^(o)  6/17 1.955^(o) 1 GT PrimerM13for(-41) CGC CAG GGT TTT CCC5/24 0.417 0.416^(o) 14/24 4.992^(o) 2 AGT CAC GAC PrimerM13rev(-27)CAG GAA ACA GCT ATG 2/17 0.235 0.236^(o)  6/17 2.006^(o) 3 ACPrimerM13rev(-48) AGC GGA TAA CAA TTT 4/23 0.348 0.326^(o)  9/233.749^(o) 4 CAC ACA GG Complement TTG GCA CTG GCC GTC 11/35  0.6290.578^(o) 20/35 10.115^(o) 5 primerM13for(-20) GTT TTA CAA CGT CGTGAC TG BJ1 CAG TCA CGA CGT TGT 5/30 0.333 0.295^(o), 13/30 4.425^(o) 6AAA ACG ACG GCC AGT 0.327^(b) 4.905^(b) BJ1EXT(mU) TTT GmUA AAA CGA CGG3/23 0.261 0.273^(b)  9/30 3.140^(b) 7 CCA GmUA AA ComplementACA ACG TCG TGA CTG 6/35 0.333 0.344^(o) 17/35 6.020^(o) 8primerM13for(-41) GGA AAA CCC TGG CGT TAC CC BJ2 GGG TAA CGC CAG GGT6/30 0.400 0.401^(o) 15/30 6.015^(o) 9 TTT CCC AGT CAC GAC 0.401^(b)6.015^(b) BJ3 GGG TAA CGC CAG GGT 5/30 0.333 0.347^(o) 15/30 5.205^(o)10 TTT CCC AGT CAC GAC 0.353^(b) 5.295^(b) BJ4 GGG TAA CGC CAG GGT 7/300.467 0.452^(o), 15/30 6.780^(o) 11 TTT TCC AGT CAC GAC 0.445^(b)6.675^(b) BJ2 TA(OMe) TTT CGC CAG GGU UUU 3/30 0.200 0.324^(a) 17/304.860^(a) 12 CCC AGU CAC GAC AAA (all 2′-OMe with the exception of Ts)BJ2 AT(OMe) AAA CGC CAG GGU UUU 3/30 0.200 0.311^(a) 17/30 4.665^(a) 13CCC AGU CAC GAC TTT (all 2′-OMe with the exception of Ts) BJ2EXT(mU)TTT CGC CAG GGmU 3/30 0.200 0.278^(b) 17/30 4.170^(b) 14mUmUmU CCC AGmU CAC GAC AAA miRNA21, RNA UAG CUU AUC AGA CUG 0/22 —0.268^(c) 11/22 2.950^(c) 15 AUG UUG A miRNA21-A15, RNAUAG CUU AUC AGA CUG 0/37 — 0.099^(o) 11/37 1.832^(o) 16 AUG UUG A₁₆Complement UCA ACA UCA GUC UGA 0/22 — 0.183^(c) 11/22 2.013^(c) 17miRNA-21, RNA UAA GCU A dmiR21 TCA ACA TCA GTC TGA 6/22 0.545 0.485^(o)11/22 5.339^(o) 18 TAA GCT A 21EXT TTT CAA CAT CAG TCT 8/24 0.6670.665^(o) 13/24 8.000^(o) 19 GAT AAG CTA dmiR21(OMe) UCA ACA UCA GUC UGA0/22 — 0.082^(b), 11/22 0.902^(b), 20 UAA GCU A (all 2'-OMe) 0.124^(c)1.365^(c) 0.26^(d) 2.85^(d) 21EXT(mU) TTT CAA CAmU CAG 3/27 0.2310.433^(a), 13/27 5.846^(a) 21 mUCmU GAmU AAG CmU 0.344^(b) 4.644^(b) AAAmiRNA122, RNA UGG AGU GUG ACA AUG 0/22 — 0.277^(c)  9/22 3.04^(c) 22GUG UUU G Complement CAA ACA CCA UUG UCA 0/22 — 0.143^(c) 13/221.573^(c) 23 miRNA122, RNA CAC UCC A dmiR122 CAA ACA CCA TTG TCA 4/220.364 0.404^(o) 13/22 4.444^(o) 24 CAC TCC A 2XdmiR122(CAA ACA CCA TTG TCA 8/44 0.364 0.380^(o) 26/44 8.36^(o) 25 CAC TCC A)₂122EXT TTT CAA ACA CCA TTG 7/25 0.560 0.602^(o) 16/25 7.525^(o) 26TCA CAC TCC A dmiR122(OMe) C AAA CAC CAU UGU CAC 0/22 — 0.088^(b), 13/220.965^(b), 27 ACU CCA (all 2'-OMe) 0.150^(c) 1.654^(c) miRNA140, RNACAG UGG UUU UAC CCU 0/22 — 0.233^(c) 13/22 2.563^(c) 28 AUG GUA GdmiR140 CTA CCA TAG GGT AAA 4/22 0.364 0.357^(o) 10/2  3.926^(o) 29ACC ACT G 2XdmiR140 (CTA CCA TAG GGT AAA 8/44 0.364 0.321^(o) 20/447.055^(o) 30 ACC ACT G)₂ 140EXT(mU) TTT CmUA CCA mUAG 3/27 0.2220.374^(a), 13/27 5.049^(a) 31 GGmU AAA ACC ACmU 0.314^(b) 4.239^(b) GAAd(CT)₁₀ CTCTCTCTCTCTCTCTCTCT 10/20  1.00 1.044^(b) 20/20 10.44^(b) 32T8, RNA (AG)₄C₂(AG)₄C₂(AG)₃CCUUC 0/32 — 0.50  9/32 8.0 33 A, foot note 4100nt RNA foot note 5  0/100 — 0.239^(c) 11.96^(c) 34 100nt RNA(OMe)foot note 6  0/100 — 0.224^(c) 11.19^(c) 35 Underlined partial sequencescorrespond to M13primers with or without a mismatch. ¹TheoreticalR(312/272) for T only equals 2x(No of T)/(total nt) (see ExperimentalSection). ²Observed R(312/272) is the observed Absorbance at 312 nmdivided by the observed absorbance at 272 nm from HPLC. ObservedR(312/272) = 2x(No of Py(OsBp)/(total nt) (see Experimental Section).³No of OsBp, on average, is determined from the equation above, and itis equal to observed R(312/272)x(total nt)/2. ^(o,a,b,c,d)are differentprotocols for preparing osmylated oligos : ^(o)batch of OsBp stocksolution prepared without dissolving bipy ahead of OsO₄ addition, 40 minwith 2.625 mM OsBp. ^(a, b, c)batch of OsBp stock solution prepared bydissolving bipy before adding OsO4; ^(a)45 min with 2.625mM OsBp; ^(b)30min with 2.625mM OsBp; ^(c)30 min with 3.94 mM OsBp. ^(d)Specialprotocol (30 min with 5.25 mM OsBp) to add about 3 OsBp per 22nt oligoin the absence of Ts, ⁴Practically 100% osmylated at all pyrimidines(Sultan M., Kanavarioti, A. (2019).). ⁵100nt RNA sequence: 5′-UUA CAGCCA CGU CUA CAG CAG UUU UAG AGC UAG AAA UAG CAA GUU AAA AUA AGG CUA GUCCGU UAU CAA CUU GAA AAA GUG GCA CCG AGU CGG UGC UUU U-3′ (SEQ ID NO:34). ⁶100nt RNA(OMe) sequence: 5′-mUmUmA CAG CCA CGU CUA CAG CAG UUU UAGAmGmC mUmAmG mAmAmA mUmAmG mCAA GUU AAA AUA AGG CUA GUC CGU UAU CAmAmCmUmU mGmAmA mAmAmA mGmUmG mGmCmA mCmCmG mAmGmU mCmGmG mUmGmC mUmUmUmU-3′; m stands for 2′-OMe (SEQ ID NO: 35).Results and Discussion

The materials used in this study were all synthetic oligos of thehighest purity and are listed in Table 1. Osmylation protocols weredeveloped by us. Intact and osmylated nucleic acids were furthercharacterized in-house by validated HPLC methods (Kanavarioti, A.(2019)). Nanopore experiments were conducted using the ONT devices andthe ONT supplied Flush buffer (ONT buffer or buffer), in addition tocompany's instructions of how to prime the flow cell, add the sample,select voltage, and acquire the raw i-t traces. No sample library wasprepared and no enzyme-assistance was exploited. Samples were preparedin 90-95% ONT buffer, unless otherwise noted. The nanopore experimentsreported here were conducted at the factory-preset, flow celltemperature in the range of 34-35° C. The raw i-t traces of all channels(a fast5 file) were captured and analyzed using OsBp_detect software(Kanavarioti, A., & Kang, A. See RNA(OsBp) event detection Pythonpackage in a public repository: hypertext transfer protocol securegithub.com/kangaroo96/osbp_detect and for step-by-step installationinstructions see here: hypertext transfer protocol securegithub.com/kangaroo96/osbp_detect/blob/master/instructions.md). Theoutput, a tsv file, is read using Microsoft Excel. It lists I₀ value foreach channel, as well as the selected events and their I_(r) value fromwhich I_(r)/I_(o) is calculated. It also lists the times, in data timepoints, of the beginning and of the end of each event (FIG. 8).OsBp_detect permits manual setting of parameters, in order to selectevents of interest (Kanavarioti, A., & Kang, A. See RNA(OsBp) eventdetection Python package in a public repository: hypertext transferprotocol secure at github.com/kangaroo96/osbp_detect and forstep-by-step installation instructions see here: hypertext transferprotocol secure atgithub.com/kangaroo96/osbp_detect/blob/master/instructions.md). Here weselected events with residence time 4≤τ≤300 data time points or theequivalent 1.3≤τ≤100 ms, and fractional residual ion currentI_(r)/I_(o)≤0.55 (FIG. 1B). FIG. 2, FIG. 4, FIG. 5, FIG. 6, and FIG. 7present histograms of count of events (abbreviated as counts or events)as a function of I_(r)/I_(o) using a 0.05 bin.

Nanopore Experiments Using the Flongle Flow Cell

FIG. 2 illustrates results exploiting the Flongle flow cell. FIG. 2A wasa first attempt to observe a hybrid. It was known from earlier work withthe MinION flow cell that T8, a 32 nt RNA with 9 pyrimidines and a totalof 8 OsBp tags (see sequence in Table 1), requires high voltage totraverse, exhibits multiple translocation events, and severely obstructsthe ionic current exhibiting a maximum of counts at(I_(r)/I_(o))max≈0.1. Repeating the experiment practically duplicatedthe earlier work on the MinION (Sultan M., Kanavarioti, A. (2019)). Eventhough not a perfect complement, d(TC)₁₀ was used to form the dscomplex, as d(CT)₁₀ can base-pair 16 out of 20 nt, including 8 GC pairs,with T8. While the experiment with probe T8 exhibited, on average, 400events per channel, the experiment using a 1:1 mixture of probe T8 andd(CT)₁₀, yielded less than 50 events per channel for some channels andzero events reported for the rest (FIG. 9A-D). FIG. 2B illustrates atest for identification of miRNA122 (Li, X-D. et al. (2017)). The probeused here is dmiR122, the exact deoxy complement of miRNA122, andcarries 4 T(OsBp) (see Table 1). The sample with probe dmiR122 aloneexhibited numerous counts, while the sample with an approximateequimolar mixture of this probe and miRNA122 exhibited markedly fewercounts. A third sample with a 4-times higher miRNA load, composed ofmiRNA21 (Thum, T. et al. (2008); Lai, J. Y. et al. (2017); Fulci, V. etal. (2007); Wang, Y. et al. (2020)) and miRNA140 (Li, X-D. et al.(2017)) also exhibited fewer counts compared to the probe sample. Thelatter suggests that identification of a target in a complex mixture ofmiRNAs is plausible. These as well as other experiments demonstrated thefeasibility of the concept presented in FIG. 1D. They also reveal thatboth the target and the probe can be either RNA or DNA, the differencebeing that probes are osmylated oligos, while targets are not. Furtherfocus was on probes that are DNA oligos, because of the lower cost andthe higher synthetic product quality compared to RNA oligos.

Alternative Methods to Test Hybridization Between a Target and its Probe

Independent means were sought to test hybridization between osmylatednucleic acids and their DNA or RNA targets. Enzymatic DNA polymeraseelongation of an unmodified primer using partially osmylated templatessM13mp18 was the first attempt to obtain support for hybridization, butelongation of primers was not detectable (data not shown). UnmodifiedssM13mp18 was then tested, as the template, and used 30 nt long,complementary T(OsBp) primers, BJ1, BJ2, BJ3, and BJ4 (see Table 1 andFIG. 11 and FIG. 13). BJ1 carries the identical sequence of primerM13for(−20) at the 3′-end, and is extended by 13 nt at the 5′-end. BJ2carries the identical sequence of primerM13 for(−41) at the 3′-end, andis extended by 6 nt at the 5′-end. BJ3 and BJ4 have the identicalsequence to BJ2 with the exception of one mismatch in the middle of thesequence. Even though BJ2, BJ3, and BJ4 carried 6, 5, and 7 T(OsBp)bases, respectively (see Table 1), they were all successfully elongated(FIG. 3A), suggesting that T(OsBp) did not prohibit 1:1 hybridizationbetween intact ssM13mp18 and the probes. In contrast, BJ1, with 6T(OsBp) did not elongate, presumably due to the presence of a T(OsBp)base at the 3′-end and the inability of the enzyme to add a nucleotideto it (FIG. 3A). Still absence of elongation with BJ1 does notnecessarily imply absence of hybridization.

These elongation experiments were done at salt concentration presumed tobe much lower than the one used for the nanopore experiments, they werelimited to ssM13mp18 and to the use of probes with sequences identicalto known primers. To extend hybridization tests to miRNAs and anyDNA/RNA oligo we developed an HPLC method as described elsewhere herein.This HPLC method is based on the HPLC method used to test for oligopurity with the following modifications: it uses (i) ONT buffer as thesample solvent and (ii) HPLC column temperature at 35° C. to mimic theONT working flow cell temperature. It should be noted that the HPLCcolumn packing may interfere with hybridization, and therefore all theHPLC-based results are purely suggestive. Having said that, there was noinstance observed where the HPLC results and the nanopore experimentcontradicted each other. To confirm hybridization HPLC analyses of threeseparate samples are required. These three samples contain (i) theprobe, (ii) the nucleic acid target molecule, and (iii) the sample withthe 1:1 mixture of the two components that contains the presumed hybrid(see FIGS. 3B-3D). Absence of hybridization is consistent with an HPLCprofile of the mixture sample that closely overlaps with the “sum” ofthe HPLC profiles of the two components (FIG. 3C). Evidence forhybridization is consistent with a hybrid peak that elutes well resolvedfrom the peaks of the target and of the probe and typically 1 to 1.5minutes later than either probe or target. In addition, the probe andhybrid peaks exhibit absorbance at 312 nm, due to the presence of theOsBp tag, but the target peak does not. 1:1 mixture samples wereprepared intentionally with a small excess of the target to prevent theprobe from being in excess. This is why in many of the HPLCchromatograms the analysis of the hybrid sample includes a smaller peakattributed to the target, in addition to the large peak attributed tothe hybrid.

FIG. 3B shows the HPLC chromatograms of samples where both oligos areunmodified nucleic acids. Here the HPLC profile of the sample with the1:1 mixture is consistent with strong hybridization, based on thefeatures discussed above. FIG. 3D is a repeat of the HPLC analyses inFIG. 3B, only that BJ2 is now osmylated; it is a probe. Thecorresponding HPLC profile of the 1:1 mixture in FIG. 3D is alsoconsistent with strong hybridization. The same HPLC method indicatedthat miRNA21 and probe 21EXT (see Table 1) do not hybridize (FIG. 3C).The absence of hybridization in FIG. 3C is attributed to the relativelylarge number of OsBp moieties present within the probe's sequence (total8 dTs, with 5 dTs found within the 21 nt sequence). Additionalhybridization tests confirmed the hypothesis that ds complex formationis prohibited in the presence of a large number of OsBp moieties on theprobe (see FIG. 10). Besides the number of OsBp moieties in a molecule,the actual location matters as well. As seen with the BJ1-4 probes,hybridization remains strong, despite a relatively large number ofT(OsBp). This is most likely because these tags occupy a small area ofthe sequence, leaving two rather long subsequences available for duplexformation with the target.

Hybridization Silences the Probe in the Presence of the DNA Oligo Target

Experiments in FIG. 2 were conducted using probe, target, and hybridconcentration in the 5 μM range. Since sample size of a MinION and thatof a Flongle flow cell is 75 μL vs. 30 μL, the 5 μM concentrationcorresponds to approximately a 0.38 vs. a 0.15 nmole sample load,respectively. It is worth mentioning that sample load for a nanoporeexperiment is not the same as sample load for an HPLC analysis, as theHPLC injection volume is typically not the same as the flow cell samplesize. FIG. 4A illustrates the HPLC hybridization test, described above,using probe BJ1 with 5 T(OsBp) and its target, complementary primer M13for(−20). The samples of probe and hybrid were used, as is, for thenanopore experiments, shown in FIG. 4B. We note that probe BJ1 was notenzymatically elongated using ssM13mp18, as the template, which weattributed to the presence of a T(OsBp) base at the 3′-end. In FIG. 4hybridization is documented both by HPLC analysis and by nanoporeexperiments, as shown by the huge drop in the number of counts reportedfor the hybrid sample compared to the counts reported with the probesample. The effect is dramatic for the (I_(r)/I_(o))max, and clearlydetectable for the rest of the I_(r)/I_(o) range.

Applied Voltage is a Critical Parameter

FIG. 4B illustrates that testing probe BJ1 with applied voltage at −180mV exhibits very low counts suggesting that probe translocation isinefficient at −180 mV. Without addition of a new sample, raising thevoltage to −220 mV yielded dramatically more counts compared to thecounts obtained at −180 mV. FIG. 11 illustrates that probes BJ2 and BJ4follow the same pattern as BJ1. This observation was attributed to thepresence of adjacent dT(OsBp) in the probes, and the notion thatdT(OsBp) exhibits the lowest observed (I_(r)/I_(o))_(max), among alltested pyrimidines, a strong indication of heavy crowding. Heavycrowding in adjacent OsBp moieties was concluded from earlier studiesusing RNAs and the MinION/CsGg (Sultan M., Kanavarioti, A. (2019)), aswell as DNAs and the α-Hemolysin nanopores (Ding, Y. & Kanavarioti, A(2016)). Since the probes are deoxyoligos and include several T bases inthe sequence, the steric hindrance is compounded. Labeled nucleic acidsmay approach the CsGg pore guided by the applied voltage drop, but totraverse the pore a certain minimum voltage is required. Multipleexperiments suggested that the applied voltage for efficienttranslocation of most of our probes via the proprietary CsGg nanopore isin the range of −210±10 mV. In contrast to the observations with theprobes, intact DNA oligos exhibit insignificant counts (Ding, Y. &Kanavarioti, A. (2016); Sultan M., Kanavarioti, A. (2019)) andtarget/unmodified miRNAs exhibit measurable counts with a slightdecreasing trend as a function of increasing voltage (FIG. 13). This isconsistent with the expectation that increased voltage results in fastertranslocation and faster translocation, in turn, results in missedevents, as the acquisition rate of the device remains constant at 3 datapoints per ms. In order to make the counts with native RNAs detectablethe total load was 4-times higher than the highest probe load.Experiments with voltage higher than −220 mV were not conducted, inorder to protect the protein pores that, in our experience, did not lastas long at −220 mV compared to −180 mV. The observation that our probespractically do not traverse the CsGg pore at −180 mV is an advantage fora diagnostic test. It provides the opportunity to deplete the samplefrom excess non-target nucleic acids at −180 mV, and then, withoutadding any a new sample, raise the voltage at −220 mV in order todetect, or not, the presence of the uncomplexed probe.

General Design for a Highly Detectable Probe

The presence of T(OsBp) moieties in the middle of a sequence is not afeature shared by many potential ctDNA or miRNA targets. Therefore,advanced probes were designed by replacing all dTs in the sequence withdU, modifying some or all the bases as 2′-OMe, added 3 adjacent dT(OsBp)at the 5′-end, and, in some cases adding 3 additional dAs at the 3′-end.Addition of dAs at the 3′-end is commonly used to facilitate pore entry(Kasianowicz, J. J., Brandin, E., Branton, D. & Deamer, D. W. (1996);Butler, T. Z., Gundlach, J. H. & Troll, M. (2007); Maglia, G., Heron, A.J., Stoddart, D., Japrung, D. & Bayley, H. (2010)). Replacing DNA baseswith 2′-OMe bases has been reported to lead to stronger hybridization(Majlessi, M., Nelson N. C. & Becker, M. M. (1998)). Replacing all dTswith dUs assures that the presence of OsBp is minimal within thesequence. This results in the lowest possible number of OsBp moietieswithin the sequence and the most unhindered hybridization with thetarget. The addition of 3 adjacent dTs at the 5′-end makes the probeundetectable at applied voltage of −180 mV and highly detectable atapplied voltage of −220 mV, as shown by the earlier experiments with theBJ1-4 probes. This probe design was then exploited in nanoporeexperiments with extra low target loads.

BJ2 TA(OMe) is a probe designed with the above features (sequence inTable 1). Hybridization between probe BJ2 TA(OMe) and the complementaryprimerM13 for(−41) was tested by HPLC at the 5 μM concentration range(FIG. 4C). FIG. 4C presents the HPLC analyses of the probe and of thehybrid samples. The target peak in the hybrid sample is easilyidentified due to its about 10% excess over the probe. The HPLC profilesat both wavelengths, 272 nm and 312 nm, are shown. Close inspection ofthese profiles illustrates that the hybrid peak has about half thecontribution at 312 nm, compared to the corresponding contribution inthe probe. This is in agreement with the expectation that the hybrid ishalf probe and half unmodified target. The actual samples tested by HPLCwere diluted with ONT buffer to produce the samples tested by nanopore.The protocol followed for this and all dilutions in this study was doneby consecutive 1:10 dilutions with ONT buffer in 0.5 mL microcentrifugetubes. The nanopore experiment with probe BJ2 TA(OMe) was done at a1000-fold dilution compared to the HPLC sample, i.e., at a 0.38 pmoleprobe load. The corresponding experiment with the hybrid was conductedat 3.75 pmole, i.e., at a 10-times higher hybrid load. The high hybridload was chosen to test the stability of the hybrid under theexperimental parameters especially under the influence of the −220 mVapplied voltage. Despite the hybrid sample being 10-times moreconcentrated than the probe, the counts obtained from the hybridexperiment were visibly fewer compared to the counts obtained from theprobe experiment and suggest that hybrid dissociation is not significantunder the tested conditions (FIG. 4D).

Hybridization silences the probe in the presence of an RNA oligo target.During the development work several probe designs were explored.Experiments with two of those designs are illustrated in FIG. 5. Probe2XdmiR122 is a 44 nt oligo with 8T(OsBp) and consists of two fuseddmiR122 (sequence in Table 1). Even though probe dmiR122 exhibitednumerous counts at −190 mV (FIG. 2B), probe 2XdmiR122 required −220 mV(FIG. 5A). The higher voltage is most likely the consequence of heavycrowding within the pore, as 2XdmiR122 incorporates two 4 nt groups with3 OsBp each within a subsequence of 26 nt. Efficient hybridizationbetween miRNA122 and 2XdmiR122 was shown by HPLC (FIG. 5C) and confirmedby nanopore, as seen by the huge drop of counts for the hybrid samplecompared to the probe sample (FIG. 5D). The advantage of such fusedsequence design lies in exploiting cases where the identical sequence ofbases is present in a longer RNA in addition to a miRNA target. Using aprobe with a design like the fused 2XdmiR122 may favor hybridizationwith the miRNA target over the long RNA target, as 2XdmiR122 can form a44 nt ds complex with miRNA122, but only a 22 nt ds complex with thelonger RNA.

FIG. 5B shows another probe design, exemplified by probe 122EXT(sequence in Table 1). Probe 122EXT has the identical sequence ofdmiR122 with the addition of 3 adjacent Ts at the 5′-end. This proberequires −220 mV to show numerous counts (solid trace), as seen bycomparison to the nanopore experiment at −180 mV (dashed trace). Ananopore experiment conducted with probe 122EXT in a sample prepared in15% human serum and 85% ONT buffer exhibits reduced counts compared tothe sample prepared in over 95% ONT buffer. The reduction in counts maybe attributed to the lower ionic strength due to the presence of serum,and/or to the aged flow cell and/or to serum interference. Despite thelower counts probe 122EXT is easily discriminated from thecontrol/buffer test indicating the unhindered probe detection in anunknown sample that contains a body fluid, such as human serum.

miRNA21 is an important biomarker for a number of diseases (Thum, T. etal. (2008); Kao, H. et al. (2017); Fulci, V. et al. (2007)), so itsidentification was tested. HPLC tests with miRNA21 and probes dmiR21(not shown) or 21EXT indicated no detectable hybridization with miRNA21(FIG. 12A, B). Inability to form the hybrid was attributed to thepresence of more than 6 T(OsBp) moieties and the fact that thesemoieties are spread over the 22 nt sequence. Advanced probe design ledto probes that efficiently hybridized with miRNA21. Probe dmiR21(OMe) isa 22 nt oligo, complementary to miRNA21, where all bases are 2′-OMe, Tsare replaced with dU, and osmylation resulted in the addition of 2.85OsBp moieties, on average, per molecule (osmylation protocol d, seeTable 1). The osmylated product is a mixture containing primarilymolecules with 2 or 3 OsBp moieties, and also molecules containing OsBpmoieties at different bases (called here topoisomers) (Sultan M.,Kanavarioti, A. (2019); Kanavarioti, A. et al. (2012)). Chromatographyresolved molecules that carry one, two or three OsBp moieties and oftenresolves topoisomers too (Kanavarioti, A. (2016). This is why the HPLCprofile of the probe consists of two separate peaks attributed tomolecules with 2 tags and to molecules with 3 tags (FIG. 6A). Similarly,the HPLC profile of the hybrid appears as multiple peaks (FIG. 6A). HPLCwas also used to test the stability of RNAs (see below) and thestability of the hybrid of dmiR21(OMe) with miRNA21 in a sample solventthat contains 15% human serum and 85% ONT buffer. FIG. 26B illustratesthat the tested RNAs, i.e., miRNA140 and the 100 nt RNA, both degradedwithin minutes, whereas the hybrid peak remained practically unchanged,suggesting that the hybrids formed between our probes and their RNAtargets are expected to be stable during the duration of an experimentin human serum.

Hybridization is consistent with the distinct HPLC profiles observedwith the probe and the hybrid samples (FIG. 6A). Because dmiR21(OMe)does not contain 3 adjacent T(OsBp), applied voltage at −180 mV wassufficient to thread this probe via the pore. Plenty of events werereported with a 0.75 nmole probe sample and markedly fewer with a 1.5nmole hybrid sample (FIG. 6B, compare solid trace with dashed trace).Counts with the hybrid appear comparable to counts obtained with buffer(not shown). Additional nanopore experiment were conducted withidentical probe and hybrid loads but in the presence of other RNAcomponents. These components were a non-target nucleic acid, miRNA140,and a 100 nt RNA, at a total 10-fold higher load compared to the probe.The nanopore profiles of the probe samples with or without the excessnon-target RNA are distinct suggesting influence by the excess materialand/or an aged flow cell. Still the two experiments in the presence ofthe non-target RNA show efficient identification of the target bycomparing the numerous counts of the probe sample to the few counts ofthe hybrid sample (second dashed line, almost indistinguishable from thefirst dashed line (hybrids)). This discrimination suggests that thepresence of non-target miRNAs and longer RNAs in a complex mixture donot prevent target identification.

Due to the importance of miRNAs as biomarkers (Li, X-D. et al. (2017);Thum, T. et al. (2008); Lai, J. Y. et al. (2015); Kao, H. et al. (2017);Fulci, V. et al. (2007); Wang, Y. et al. (2020)), experiments wereconducted at extra low load with a probe design that is broadlyapplicable to any target sequence and has optimal translocationproperties. Probes 140EXTmU and 21EXTmU to target miRNA140 and miRNA21,respectively, were selected. These probes have a sequence complementaryto their target, mU replacing T within the sequence, 3 additionaladjacent dTs at the 5′-end, and 2 or 3 additional dAs at the 3′-end.These oligos were osmylated using the validated labeling process thatadds, on average, 4 to 5 OsBp tags per molecule, 3 of which occupy the5′-end, and the other 1 or 2 are randomly allocated within the sequence(see Table 1). Due to the heavy crowding at the one end of the probe,applied voltage in the range of −210±10 mV is required for probeefficient translocation and detection (per discussion above). FIG. 7Aillustrates detection with excellent sensitivity at the 47 fmole levelwith 140EXT(mU), and FIG. 7B illustrates detection of this probe at aneven higher sensitivity of 3.5 amole probe load. Identification of thetarget miRNA140 is also evident by comparing the probe to the hybridwith hybrid load also at 3.5 amole. FIG. 7C shows the HPLC profile ofthe hybrid sample between probe 21EXT(mU) and miRNA21, which was thendiluted by a 3×10⁸ factor for the nanopore experiment to test the hybridat the 2.7 amole level (FIG. 7D). The 21EXT(mU) probe sample (HPLCprofile not shown) was diluted by a 1×10⁹ factor in order to conduct thenanopore experiment at the 0.9 amole level of (FIG. 7D) which is thelowest load tested in this study. Identification of miRNA21 is evidentin FIG. 7D by visual comparison of the probe counts to the counts of thehybrid. The reason that the counts observed with the 140EXT(mU) probe(top, reaching 6,000) are fewer compared to the counts observed with the21EXT(mU) probe (bottom, reaching 24,000) is, at least partially, due toan aged flow cell that had only about 30% of working pores. Whether ornot proportionality in probe counts can be obtained from the ONT/OsBpplatform for a specific probe was not tested here, primarily becauseprobe concentration is a known quantity. Most importantly,identification of the target is based on a nanopore profile thatexhibits an insignificant number of counts, comparable to the counts ofthe control/buffer experiment. The main reason we tested every probe bynanopore, is because we wanted to compare different probe designs andconfirm high detectability and high sensitivity. The potential of theONT/OsBp nanopore platform

The above experiments range in probe load from 0.38 nmole to 0.9 amole,practically spanning 9 orders of magnitude. In this range evidence ispresented for probe detection, and clear distinction between the samplethat contains the target and the sample that doesn't. A lowerdetectability limit is proposed as 3-times higher count of events withprobe alone compared to the counts of the hybrid. Proportionalitybetween the count of events and the probe concentration was not testedhere, as the duration of the experiments remains in the range of 1 to 3h and does not reflect the sample load variation. Since our test doesnot detect the hybrid and therefore does not measure the hybridmolecules, quantification of the target depends on the known probe load.The test requires an estimate for target load and depending on theoutcome of the first experiment, one can vary the probe load by a factorof 5 below or above in the next experiment. It is estimated that incertain aspects, target quantification can be attained with about 30%accuracy. A number of representative probe designs were explored and onedesign proposed that can practically match any ssDNA or ssRNA oligotarget. This type of probe, with examples identified as 140EXT(mU) and21EXT(mU), exhibited high sensitivity at the amole load level. Noattempt was made to test for specificity. Discrimination between onetarget and another target with similar sequence will need to beaddressed case-by-case. The HPLC method developed here can evaluatehybridization between a target and a tentative probe and it can alsoassess probe hybridization extent and tentative discrimination betweentwo targets with similar sequence. Here the flow cell temperature was atthe factory preset temperature, but nanopore proteins are known to bestable in a certain temperature range. Flow cell temperature, if left atthe discretion of the user, can provide a means to improve specificity.We also did not replace the proprietary ONT Flush buffer. The latter wasdeveloped for sequencing, and a different buffer may be developed to bemore suitable for other applications including the present invention.

Preliminary experiments indicated that the hybrid of miRNA21 withdmiR21(OMe) is relatively stable in a 15%-85% serum-ONT buffer and thatprobe 122EXT is detectable by nanopore in a 15%-85% serum-ONT buffer.These experiments suggest that using the ONT/OsBp nanopore platform withblood serum samples is feasible. Considering that the MinION flow celluses 75 μL sample volume and 15% could be blood serum, thenapproximately 114 of a human serum sample can be directly tested in ananopore experiment. Assuming that an 11 μL human serum sample containsabout 3 amole of miRNA21, miRNA140, or tentatively any other miRNA, aprobe designed according the present invention should be able to detectit, and as a corollary detect the presence/absence of the target. Inthis context, aspects of the invention qualify as a follow-up,point-of-care diagnostic test.

The breadth and scope of the present disclosure should not be limited byany of the above-described exemplary embodiments, but should be definedonly in accordance with the following claims and their equivalents.

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MSDS and Information is obtained from the following link to UCLA    Chemisty Department, see    https://www.chemistry.ucla.edu/sites/default/files/safety/sop/SOP_Osmium_Tetroxide.pdf

What is claimed is:
 1. A method for detecting the presence of a nucleicacid target molecule in a biological sample, the method comprising thesteps of: (a) contacting a test sample that comprises (i) a biologicalsample comprising a nucleic acid target molecule and (ii) an osmylatedsingle-stranded oligonucleotide probe comprising at least one pyrimidineresidue covalently bonded to a substituted or unsubstituted Osmiumtetroxide (OsO₄)-2,2′-bypyridine group (OsBp group), wherein thesequence of the probe is at least partially complementary to thesequence of the nucleic acid target molecule, to allow the formation ofa hybridized probe/target complex, wherein at least one osmylatedpyrimidine residue is a thymidine residue (T); (b) using a nanoporedevice to detect in the test sample the number of events whereinunhybridized osmylated-probe traverses the nanopore; and (c)(i)comparing the number of events detected in the test sample to a numberof corresponding probe sample events wherein unhybridizedosmylated-probe traverses the nanopore in the absence of the nucleicacid target molecule, wherein a reduction in the number of eventsdetected in the test sample relative to the number of probe sampleevents is indicative of the formation of the hybridized probe/targetcomplex in step (a) and the presence of the nucleic acid target moleculein the test sample; (c)(ii) comparing the number of events detected inthe test sample to the noise of a corresponding baseline sample thatdoes not contain any osmylated-probe, wherein an absence of an increasein the number of events detected in the test sample relative to thenoise of the baseline sample is indicative of the formation of thehybridized probe/target complex in step (a) and the presence of thenucleic acid target molecule in the test sample; and/or (c)(iii)comparing the number of events detected in the test sample to a numberof corresponding control sample events wherein unhybridizedosmylated-probe traverses the nanopore in the presence of a known amountof the nucleic acid target molecule, wherein a reduction in the numberof events detected in the test sample relative to the number of controlsample events is indicative of increased formation of the hybridizedprobe/target complex in step (a) and the presence of a higher amount ofthe nucleic acid target molecule in the test sample over the controlsample or wherein an increase in the number of events detected in thetest sample relative to the number of control sample events using thesame amount of probe is indicative more unhybridized probe and thus of alower amount of the nucleic acid target molecule in the test samplecompared to the control sample.
 2. The method of claim 1 comprising thestep of: (c)(i) comparing the number of events detected in the testsample to a number of corresponding probe sample events whereinunhybridized osmylated-probe traverses the nanopore in the absence ofthe nucleic acid target molecule, wherein a reduction in the number ofevents detected in the test sample relative to the number of probesample events is indicative of the formation of the hybridizedprobe/target complex in step (a) and the presence of the nucleic acidtarget molecule in the test sample; and/or (c)(ii) comparing the numberof events detected in the test sample to the noise of a correspondingbaseline sample that does not contain any osmylated-probe, wherein anabsence of an increase in the number of events detected in the testsample relative to the noise of the baseline sample is indicative of theformation of the hybridized probe/target complex in step (a) and thepresence of the nucleic acid target molecule in the test sample.
 3. Themethod of claim 1 comprising the step of (c)(i) comparing the number ofevents detected in the test sample to a number of corresponding probesample events wherein unhybridized osmylated-probe traverses thenanopore in the absence of the nucleic acid target molecule, wherein areduction in the number of events detected in the test sample relativeto the number of probe sample events is indicative of the formation ofthe hybridized probe/target complex in step (a) and the presence of thenucleic acid target molecule in the test sample.
 4. The method of claim3, wherein the number of corresponding probe sample events is the numberof events contemporaneously detected in one or more probe samples at thetime of detection of the number of events in the test sample and/or is apredetermined value for a given amount of probe.
 5. The method of claim1 comprising the step of (c)(ii) comparing the number of events detectedin the test sample to the noise of a corresponding baseline sample thatdoes not contain any osmylated-probe, wherein an absence of an increasein the number of events detected in the test sample relative to thenoise of the baseline sample is indicative of the formation of thehybridized probe/target complex in step (a) and the presence of thenucleic acid target molecule in the test sample.
 6. The method of claim5, wherein the noise of a corresponding baseline sample iscontemporaneously determined in one or more baseline samples at the timeof detection of the number of events in the test sample and/or is apredetermined noise value for a certain composition of baseline sample.7. The method of claim 1 comprising the step of (c)(iii) comparing thenumber of events detected in the test sample to a number ofcorresponding control sample events wherein unhybridized osmylated-probetraverses the nanopore in the presence of a known amount of the nucleicacid target molecule, wherein a reduction in the number of eventsdetected in the test sample relative to the number of control sampleevents is indicative of increased formation of the hybridizedprobe/target complex in step (a) and the presence of a higher amount ofthe nucleic acid target molecule in the test sample over the controlsample or wherein an increase in the number of events detected in thetest sample relative to the number of control sample events using thesame amount of probe is indicative more unhybridized probe and thus of alower amount of the nucleic acid target molecule in the test samplecompared to the control sample.
 8. The method of claim 7, wherein thenumber of corresponding control sample events is the number of eventscontemporaneously detected in one or more control samples at the time ofdetection of the number of events in the test sample and/or is apredetermined value for a given amount of probe mixed with a givenamount nucleic acid target molecule.
 9. The method of claim 1, whereinthe amount of the probe in the test sample is about equal to or lessthan the amount of the nucleic acid target molecule in the test sample.10. The method of claim 1, wherein the 2,2′-bipyridine in the OsBp groupis substituted.
 11. The method of claim 10, wherein the 2,2′-bipyridinein the OsBp group is substituted with one or more methyl or ethylgroups.
 12. The method of claim 1, wherein: (i) the probe is DNA, andoptionally wherein at least one of the sugars in the nucleic acidbackbone are 2′-OMe-deoxyribose; or (ii) the probe is RNA, andoptionally wherein at least one of the sugars in the nucleic acidbackbone are 2′-OMe-ribose.
 13. The method of claim 1, wherein theosmylated probe has a length of about 12 to 50 nucleotides.
 14. Themethod of claim 1, wherein the nucleic acid target molecule is acirculating tumor DNA (ctDNA), cell-free DNA (cfDNA), miRNA, afragmented coding RNA, or a non-coding RNA.
 15. The method of claim 1,wherein the osmylated probe comprises at least two, three, four, five,or six osmylated pyrimidine residues and/or, the osmylated probecomprises one, two, three, four, five, or six adenosine residues (dA orA) at the 5′-end or 3′-end of the probe.
 16. The method of claim 15,wherein the osmylated probe comprises at least two, three, or fouradjacent osmylated pyrimidine residues.
 17. The method of claim 15,wherein the osmylated probe comprises at least two, three, four, five,or six osmylated thymidine residues (T).
 18. The method of claim 17,wherein the osmylated probe comprises at least two, three, four adjacentosmylated thymidine residues (T).
 19. The method of claim 15, whereinone or more of said 5′-end or 3′-end adenosine residues does nothybridize to the target nucleic acid target molecule.
 20. The method ofclaim 19, wherein none of said 5′-end or 3′-end adenosine residues (dAor A) hybridizes to the nucleic acid target molecule.
 21. The method ofclaim 1, wherein: (i) the osmylated probe comprises at least two, three,or four adjacent osmylated pyrimidine residues located at the 5′-end or3′-end of the probe and/or, (ii) the osmylated probe does not comprisetwo or more adjacent osmylated pyrimidine residues that are not locatedat either the 5′-end or 3′-end of the probe.
 22. The method of claim 21,wherein (i) the osmylated probe comprises at least two, three, or fouradjacent osmylated pyrimidine residues located at the 5′-end or 3′-endof the probe and one or more of said 5′-end or 3′-end pyrimidineresidues does not hybridize to the nucleic acid target molecule.
 23. Themethod of claim 22, wherein (i) the osmylated probe comprises at leasttwo adjacent osmylated pyrimidine residues located at the 5′-end or3′-end of the probe and none of said 5′-end or 3′-end pyrimidineresidues hybridizes to the nucleic acid target molecule.
 24. The methodof claim 21, wherein (ii) the osmylated probe does not comprise two ormore adjacent osmylated thymidine residues (T) that are not located ateither the 5′-end or 3′-end of the probe.
 25. The method of claim 21,wherein (i) the osmylated probe comprises at least two, three, or fouradjacent osmylated thymidine residues (T) located at the 5′-end or3′-end of the probe and one or more of said 5′-end or 3′-end thymidineresidues (T) does not hybridize to the nucleic acid target molecule. 26.The method of claim 25, wherein (i) the osmylated probe comprises atleast two adjacent osmylated thymidine residues (T) located at the5′-end or 3 ‘-end of the probe and none of said 5’-end or 3′-endthymidine residues (T) hybridizes to the nucleic acid target molecule.27. The method of claim 1, wherein at least about 95% of the pyrimidineresidues, within the sequence of the oligonucleotide probe moleculewhich is complementary to the nucleic acid target molecule are notosmylated.
 28. The method of claim 1, wherein at least about 95% of thethymidine residues (T) in the oligonucleotide probe molecule areosmylated, and/or, wherein at least about 80% of pyrimidines present inthe probe, other than thymidine (T), are not osmylated.
 29. The methodof claim 28, wherein at least about 95% of the thymidine residues (T) inthe oligonucleotide probe molecule are osmylated and wherein at leastabout 80% of pyrimidines present in the probe, other than thymidine (T),are not osmylated.
 30. The method of claim 1, wherein the probe is DNAbut wherein at least one thymidine residue (T) in the probe sequence,other than adjacent thymidine residues (T) located at the 5′-end or3′-end, is replaced by a uridine (U), a 2′-OMeU (mU), or a deoxyuridine(dU) residue.
 31. The method of claim 30, wherein at least about 80% ofthe thymidine residues (T) in the probe sequence, other than adjacentthymidine residues (T) located at the 5′-end or 3′-end, are replaced byuridine (U), 2′-OMeU (mU), or deoxyuridine (dU) residues.
 32. The methodof claim 1, wherein the nanopore device allows the voltage-driventranslocation of osmylated and non-osmylated single-stranded nucleicacids but prevents the translocation of double-stranded nucleic acids.33. The method of claim 32, wherein a voltage of at least about −180 mVis applied to detect the presence of the nucleic acid target molecule.34. The method of claim 33, wherein a voltage of between about −180 mVand about −250 mV is applied to detect the presence of the nucleic acidtarget molecule.
 35. The method of claim 33, wherein a voltage of atleast about −180 mV is applied to detect the presence of the target anda voltage of less than about −200 mV is applied before the voltageapplied to detect the presence of the nucleic acid target molecule. 36.The method of claim 35, wherein a voltage of between about −180 mV andabout −250 mV is applied to detect the presence of the target and avoltage of less than about −200 mV is applied before the voltage appliedto detect the presence of the nucleic acid target molecule.
 37. Themethod of claim 1, wherein the nanopore device allows for thedistinction between different osmylated probes and/or the multiplexdetection of multiple different nucleic acid target molecules in a testsample.
 38. The method of claim 1, further comprising detecting anamount of the nucleic acid target molecule, wherein the detecting of themethod can detect an amount of less than about 1 pM of the nucleic acidtarget molecule in the test sample.
 39. The method of claim 38, whereinthe detecting of the method can detect an amount of between about 0.1 aMand about 1 pM of the nucleic acid target molecule in the test sample.40. The method of claim 39, wherein the detecting of the method candetect an amount of between about 1 aM and about 1 pM of the nucleicacid target molecule in the test sample.